WO2010025424A1 - Methods and compositions for diagnosis and prognosis of renal injury and renal failure - Google Patents

Methods and compositions for diagnosis and prognosis of renal injury and renal failure Download PDF

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Publication number
WO2010025424A1
WO2010025424A1 PCT/US2009/055449 US2009055449W WO2010025424A1 WO 2010025424 A1 WO2010025424 A1 WO 2010025424A1 US 2009055449 W US2009055449 W US 2009055449W WO 2010025424 A1 WO2010025424 A1 WO 2010025424A1
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Prior art keywords
renal
measured concentration
cohort
subject
future
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PCT/US2009/055449
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French (fr)
Inventor
Joseph Anderberg
Jeff Gray
Paul Mcpherson
Kevin Nakamura
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Astute Medical, Inc.
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Publication date
Priority to CN200980140805.3A priority Critical patent/CN102187220B/en
Application filed by Astute Medical, Inc. filed Critical Astute Medical, Inc.
Priority to NZ591437A priority patent/NZ591437A/en
Priority to JP2011525260A priority patent/JP2012501456A/en
Priority to MX2013010617A priority patent/MX341926B/en
Priority to BRPI0917711A priority patent/BRPI0917711A2/en
Priority to MX2011002233A priority patent/MX2011002233A/en
Priority to EP09810695.8A priority patent/EP2324355B1/en
Priority to CA2735587A priority patent/CA2735587A1/en
Priority to US13/061,413 priority patent/US11150250B2/en
Priority to AU2009285550A priority patent/AU2009285550B2/en
Publication of WO2010025424A1 publication Critical patent/WO2010025424A1/en
Priority to HK11107251.6A priority patent/HK1153268A1/en
Priority to US17/473,710 priority patent/US20230020055A1/en

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/34—Genitourinary disorders
    • G01N2800/347—Renal failures; Glomerular diseases; Tubulointerstitial diseases, e.g. nephritic syndrome, glomerulonephritis; Renovascular diseases, e.g. renal artery occlusion, nephropathy

Definitions

  • the kidney is responsible for water and solute excretion from the body. Its functions include maintenance of acid-base balance, regulation of electrolyte concentrations, control of blood volume, and regulation of blood pressure. As such, loss of kidney function through injury and/or disease results in substantial morbidity and mortality. A detailed discussion of renal injuries is provided in Harrison's Principles of Internal Medicine, 17 th Ed., McGraw Hill, New York, pages 1741-1830, which are hereby incorporated by reference in their entirety. Renal disease and/or injury may be acute or chronic.
  • Acute and chronic kidney disease are described as follows (from Current Medical Diagnosis & Treatment 2008, 47 th Ed, McGraw Hill, New York, pages 785-815, which are hereby incorporated by reference in their entirety): "Acute renal failure is worsening of renal function over hours to days, resulting in the retention of nitrogenous wastes (such as urea nitrogen) and creatinine in the blood. Retention of these substances is called azotemia.
  • Chronic renal failure chronic kidney disease results from an abnormal loss of renal function over months to years”.
  • Acute renal failure also known as acute kidney injury, or AKI
  • AKI acute kidney injury
  • ARF nitrogenous (urea and creatinine) and non- nitrogenous waste products that are normally excreted by the kidney, a reduction in urine output, or both. It is reported that ARF complicates about 5% of hospital admissions, 4-15% of cardiopulmonary bypass surgeries, and up to 30% of intensive care admissions.
  • ARF may be categorized as prerenal, intrinsic renal, or postrenal in causation.
  • Intrinsic renal disease can be further divided into glomerular, tubular, interstitial, and vascular abnormalities.
  • Major causes of ARF are described in the following table, which is adapted from the Merck Manual, 17 th ed., Chapter 222, and which is hereby incorporated by reference in their entirety:
  • ischemic ARF the course of the disease may be divided into four phases.
  • an initiation phase which lasts hours to days, reduced perfusion of the kidney is evolving into injury. Glomerular ultrafiltration reduces, the flow of filtrate is reduced due to debris within the tubules, and back leakage of filtrate through injured epithelium occurs.
  • Renal injury can be mediated during this phase by reperfusion of the kidney.
  • Initiation is followed by an extension phase which is characterized by continued ischemic injury and inflammation and may involve endothelial damage and vascular congestion.
  • the maintenance phase lasting from 1 to 2 weeks, renal cell injury occurs, and glomerular filtration and urine output reaches a minimum.
  • a recovery phase can follow in which the renal epithelium is repaired and GFR gradually recovers. Despite this, the survival rate of subjects with ARF may be as low as about 60%.
  • Acute kidney injury caused by radiocontrast agents also called contrast media
  • other nephrotoxins such as cyclosporine, antibiotics including aminoglycosides and anticancer drugs such as cisplatin manifests over a period of days to about a week.
  • Contrast induced nephropathy (CIN, which is AKI caused by radiocontrast agents) is thought to be caused by intrarenal vasoconstriction (leading to ischemic injury) and from the generation of reactive oxygen species that are directly toxic to renal tubular epithelial cells.
  • CIN classically presents as an acute (onset within 24-48h) but reversible (peak 3-5 days, resolution within 1 week) rise in blood urea nitrogen and serum creatinine.
  • a commonly reported criteria for defining and detecting AKI is an abrupt (typically within about 2-7 days or within a period of hospitalization) elevation of serum creatinine.
  • serum creatinine elevation to define and detect AKI is well established, the magnitude of the serum creatinine elevation and the time over which it is measured to define AKI varies considerably among publications.
  • relatively large increases in serum creatinine such as 100%, 200%, an increase of at least 100% to a value over 2 mg/dL and other definitions were used to define AKI.
  • the recent trend has been towards using smaller serum creatinine rises to define AKI.
  • “Failure” serum creatinine increased 3.0 fold from baseline OR creatinine >355 ⁇ mol/1 (with a rise of >44) or urine output below 0.3 ml/kg/hr for 24 h or anuria for at least 12 hours;
  • ESRD end stage renal disease — the need for dialysis for more than 3 months.
  • RIFLE criteria which provide a useful clinical tool to classify renal status.
  • the RIFLE criteria provide a uniform definition of AKI which has been validated in numerous studies.
  • Stage I increase in serum creatinine of more than or equal to 0.3 mg/dL (> 26.4 ⁇ mol/L) or increase to more than or equal to 150% (1.5-fold) from baseline OR urine output less than 0.5 mL/kg per hour for more than 6 hours;
  • Stage II increase in serum creatinine to more than 200% (> 2-fold) from baseline OR urine output less than 0.5 mL/kg per hour for more than 12 hours
  • Stage III increase in serum creatinine to more than 300% (> 3-fold) from baseline OR serum creatinine > 354 ⁇ mol/L accompanied by an acute increase of at least 44 ⁇ mol/L OR urine output less than 0.3 mL/kg per hour for 24 hours or anuria for 12 hours.
  • the CIN Consensus Working Panel uses a serum creatinine rise of 25% to define Contrast induced nephropathy (which is a type of AKI).
  • Contrast induced nephropathy which is a type of AKI.
  • the consensus is that small changes in serum creatinine, such as 0.3 mg/dL or 25%, are sufficient to detect AKI (worsening renal function) and that the magnitude of the serum creatinine change is an indicator of the severity of the AKI and mortality risk.
  • serum creatinine is generally regarded to have several limitations in the diagnosis, assessment and monitoring of AKI patients.
  • the time period for serum creatinine to rise to values (e.g., a 0.3 mg/dL or 25% rise) considered diagnostic for AKI can be 48 hours or longer depending on the definition used. Since cellular injury in AKI can occur over a period of hours, serum creatinine elevations detected at 48 hours or longer can be a late indicator of injury, and relying on serum creatinine can thus delay diagnosis of AKI.
  • serum creatinine is not a good indicator of the exact kidney status and treatment needs during the most acute phases of AKI when kidney function is changing rapidly. Some patients with AKI will recover fully, some will need dialysis (either short term or long term) and some will have other detrimental outcomes including death, major adverse cardiac events and chronic kidney disease. Because serum creatinine is a marker of filtration rate, it does not differentiate between the causes of AKI (pre-renal, intrinsic renal, post-renal obstruction, atheroembolic, etc) or the category or location of injury in intrinsic renal disease (for example, tubular, glomerular or interstitial in origin). Urine output is similarly limited, Knowing these things can be of vital importance in managing and treating patients with AKI.
  • measurement of one or more markers selected from the group consisting of soluble p-selectin, protein NOV homolog, soluble epidermal growth factor receptor, netrin-4, haptoglobin, heat shock protein beta-1, alpha- 1 -antitrypsin, leukocyte elastase, soluble tumor necrosis factor receptor superfamily member 6, soluble tumor necrosis factor ligand superfamily member 6, soluble intercellular adhesion molecule 2, caspase-3 (and most preferably active caspase-3), and soluble platelet endothelial cell adhesion molecule (collectively referred to herein as "kidney injury markers, and individually as a “kidney injury marker”) can be used for diagnosis, prognosis, risk stratification, staging, monitoring, categorizing and determination of further diagnosis and treatment regimens in subjects suffering or at risk of suffering from an injury to renal function, reduced renal function, and
  • kidney injury markers may be used, individually or in panels comprising a plurality of kidney injury markers, for risk stratification (that is, to identify subjects at risk for a future injury to renal function, for future progression to reduced renal function, for future progression to ARF, for future improvement in renal function, etc.); for diagnosis of existing disease (that is, to identify subjects who have suffered an injury to renal function, who have progressed to reduced renal function, who have progressed to ARF, etc.); for monitoring for deterioration or improvement of renal function; and for predicting a future medical outcome, such as improved or worsening renal function, a decreased or increased mortality risk, a decreased or increased risk that a subject will require renal replacement therapy (i.e., hemodialysis, peritoneal dialysis, hemofiltration, and/or renal transplantation, a decreased or increased risk that a subject will recover from an injury to renal function, a decreased or increased risk that a subject will recover from ARF, a decreased or increased risk that a subject will progress to end stage renal disease,
  • the present invention relates to methods for evaluating renal status in a subject. These methods comprise performing an assay method that is configured to detect one or more kidney injury markers of the present invention in a body fluid sample obtained from the subject.
  • the assay result(s) for example a measured concentration of one or more markers selected from the group consisting of soluble p-selectin, protein NOV homolog, soluble epidermal growth factor receptor, netrin-4, haptoglobin, heat shock protein beta-1, alpha- 1 -antitrypsin, leukocyte elastase, soluble tumor necrosis factor receptor superfamily member 6, soluble tumor necrosis factor ligand superfamily member 6, soluble intercellular adhesion molecule 2, caspase-3 (and most preferably active caspase-3), and soluble platelet endothelial cell adhesion molecule is/are then correlated to the renal status of the subject.
  • This correlation to renal status may include correlating the assay result(s) to one or more of risk stratification, diagnosis, prognosis, staging, classifying and monitoring of the subject as described herein.
  • the present invention utilizes one or more kidney injury markers of the present invention for the evaluation of renal injury.
  • the methods for evaluating renal status described herein are methods for risk stratification of the subject; that is, assigning a likelihood of one or more future changes in renal status to the subject.
  • the assay result(s) is/are correlated to one or more such future changes. The following are preferred risk stratification embodiments.
  • these methods comprise determining a subject's risk for a future injury to renal function, and the assay result(s) is/are correlated to a likelihood of such a future injury to renal function.
  • the measured concentration(s) may each be compared to a threshold value.
  • a threshold value For a "positive going" kidney injury marker, an increased likelihood of suffering a future injury to renal function is assigned to the subject when the measured concentration is above the threshold, relative to a likelihood assigned when the measured concentration is below the threshold.
  • a "negative going" kidney injury marker an increased likelihood of suffering a future injury to renal function is assigned to the subject when the measured concentration is below the threshold, relative to a likelihood assigned when the measured concentration is above the threshold.
  • these methods comprise determining a subject's risk for future reduced renal function, and the assay result(s) is/are correlated to a likelihood of such reduced renal function.
  • the measured concentrations may each be compared to a threshold value.
  • a threshold value For a "positive going" kidney injury marker, an increased likelihood of suffering a future reduced renal function is assigned to the subject when the measured concentration is above the threshold, relative to a likelihood assigned when the measured concentration is below the threshold.
  • a "negative going" kidney injury marker an increased likelihood of future reduced renal function is assigned to the subject when the measured concentration is below the threshold, relative to a likelihood assigned when the measured concentration is above the threshold.
  • these methods comprise determining a subject's likelihood for a future improvement in renal function, and the assay result(s) is/are correlated to a likelihood of such a future improvement in renal function.
  • the measured concentration(s) may each be compared to a threshold value.
  • a threshold value For a "positive going" kidney injury marker, an increased likelihood of a future improvement in renal function is assigned to the subject when the measured concentration is below the threshold, relative to a likelihood assigned when the measured concentration is above the threshold.
  • a "negative going" kidney injury marker an increased likelihood of a future improvement in renal function is assigned to the subject when the measured concentration is above the threshold, relative to a likelihood assigned when the measured concentration is below the threshold.
  • these methods comprise determining a subject's risk for progression to ARF, and the result(s) is/are correlated to a likelihood of such progression to ARF.
  • the measured concentration(s) may each be compared to a threshold value.
  • a threshold value For a "positive going" kidney injury marker, an increased likelihood of progression to ARF is assigned to the subject when the measured concentration is above the threshold, relative to a likelihood assigned when the measured concentration is below the threshold.
  • a "negative going" kidney injury marker an increased likelihood of progression to ARF is assigned to the subject when the measured concentration is below the threshold, relative to a likelihood assigned when the measured concentration is above the threshold.
  • these methods comprise determining a subject's outcome risk, and the assay result(s) is/are correlated to a likelihood of the occurrence of a clinical outcome related to a renal injury suffered by the subject. For example, the measured concentration(s) may each be compared to a threshold value.
  • kidney injury marker For a "positive going" kidney injury marker, an increased likelihood of one or more of: acute kidney injury, progression to a worsening stage of AKI, mortality, a requirement for renal replacement therapy, a requirement for withdrawal of renal toxins, end stage renal disease, heart failure, stroke, myocardial infarction, progression to chronic kidney disease, etc., is assigned to the subject when the measured concentration is above the threshold, relative to a likelihood assigned when the measured concentration is below the threshold.
  • kidney injury marker For a "negative going" kidney injury marker, an increased likelihood of one or more of: acute kidney injury, progression to a worsening stage of AKI, mortality, a requirement for renal replacement therapy, a requirement for withdrawal of renal toxins, end stage renal disease, heart failure, stroke, myocardial infarction, progression to chronic kidney disease, etc., is assigned to the subject when the measured concentration is below the threshold, relative to a likelihood assigned when the measured concentration is above the threshold.
  • the likelihood or risk assigned is that an event of interest is more or less likely to occur within 180 days of the time at which the body fluid sample is obtained from the subject.
  • the likelihood or risk assigned relates to an event of interest occurring within a shorter time period such as 18 months, 120 days, 90 days, 60 days, 45 days, 30 days, 21 days, 14 days, 7 days, 5 days, 96 hours, 72 hours, 48 hours, 36 hours, 24 hours, 12 hours, or less.
  • a risk at 0 hours of the time at which the body fluid sample is obtained from the subject is equivalent to diagnosis of a current condition.
  • the subject is selected for risk stratification based on the pre-existence in the subject of one or more known risk factors for prerenal, intrinsic renal, or postrenal ARF.
  • a subject undergoing or having undergone major vascular surgery, coronary artery bypass, or other cardiac surgery a subject having pre-existing congestive heart failure, preeclampsia, eclampsia, diabetes mellitus, hypertension, coronary artery disease, proteinuria, renal insufficiency, glomerular filtration below the normal range, cirrhosis, serum creatinine above the normal range, or sepsis; or a subject exposed to NSAIDs, cyclosporines, tacrolimus, aminoglycosides, foscarnet, ethylene glycol, hemoglobin, myoglobin, ifosfamide, heavy metals, methotrexate, radiopaque contrast agents, or streptozotocin are all preferred subjects for monitoring risks according to
  • pre- existence in this context is meant that the risk factor exists at the time the body fluid sample is obtained from the subject.
  • a subject is chosen for risk stratification based on an existing diagnosis of injury to renal function, reduced renal function, or ARF.
  • the methods for evaluating renal status described herein are methods for diagnosing a renal injury in the subject; that is, assessing whether or not a subject has suffered from an injury to renal function, reduced renal function, or ARF.
  • the assay result(s) for example a measured concentration of one or more markers selected from the group consisting of soluble p-selectin, protein NOV homolog, soluble epidermal growth factor receptor, netrin-4, haptoglobin, heat shock protein beta-1, alpha- 1 -antitrypsin, leukocyte elastase, soluble tumor necrosis factor receptor superfamily member 6, soluble tumor necrosis factor ligand superfamily member 6, soluble intercellular adhesion molecule 2, caspase-3 (and most preferably active caspase-3), and soluble platelet endothelial cell adhesion molecule is/are correlated to the occurrence or nonoccurrence of a change in renal status.
  • the following are preferred diagnostic embodiments.
  • these methods comprise diagnosing the occurrence or nonoccurrence of an injury to renal function, and the assay result(s) is/are correlated to the occurrence or nonoccurrence of such an injury.
  • each of the measured concentration(s) may be compared to a threshold value.
  • an increased likelihood of the occurrence of an injury to renal function is assigned to the subject when the measured concentration is above the threshold (relative to the likelihood assigned when the measured concentration is below the threshold); alternatively, when the measured concentration is below the threshold, an increased likelihood of the nonoccurrence of an injury to renal function may be assigned to the subject (relative to the likelihood assigned when the measured concentration is above the threshold).
  • an increased likelihood of the occurrence of an injury to renal function is assigned to the subject when the measured concentration is below the threshold (relative to the likelihood assigned when the measured concentration is above the threshold); alternatively, when the measured concentration is above the threshold, an increased likelihood of the nonoccurrence of an injury to renal function may be assigned to the subject (relative to the likelihood assigned when the measured concentration is below the threshold).
  • these methods comprise diagnosing the occurrence or nonoccurrence of reduced renal function, and the assay result(s) is/are correlated to the occurrence or nonoccurrence of an injury causing reduced renal function.
  • each of the measured concentration(s) may be compared to a threshold value.
  • an increased likelihood of the occurrence of an injury causing reduced renal function is assigned to the subject when the measured concentration is above the threshold (relative to the likelihood assigned when the measured concentration is below the threshold); alternatively, when the measured concentration is below the threshold, an increased likelihood of the nonoccurrence of an injury causing reduced renal function may be assigned to the subject (relative to the likelihood assigned when the measured concentration is above the threshold).
  • an increased likelihood of the occurrence of an injury causing reduced renal function is assigned to the subject when the measured concentration is below the threshold (relative to the likelihood assigned when the measured concentration is above the threshold); alternatively, when the measured concentration is above the threshold, an increased likelihood of the nonoccurrence of an injury causing reduced renal function may be assigned to the subject (relative to the likelihood assigned when the measured concentration is below the threshold).
  • these methods comprise diagnosing the occurrence or nonoccurrence of ARF, and the assay result(s) is/are correlated to the occurrence or nonoccurrence of an injury causing ARF.
  • each of the measured concentration(s) may be compared to a threshold value.
  • an increased likelihood of the occurrence of ARF is assigned to the subject when the measured concentration is above the threshold (relative to the likelihood assigned when the measured concentration is below the threshold); alternatively, when the measured concentration is below the threshold, an increased likelihood of the nonoccurrence of ARF may be assigned to the subject (relative to the likelihood assigned when the measured concentration is above the threshold).
  • an increased likelihood of the occurrence of ARF is assigned to the subject when the measured concentration is below the threshold (relative to the likelihood assigned when the measured concentration is above the threshold); alternatively, when the measured concentration is above the threshold, an increased likelihood of the nonoccurrence of ARF may be assigned to the subject (relative to the likelihood assigned when the measured concentration is below the threshold).
  • these methods comprise diagnosing a subject as being in need of renal replacement therapy, and the assay result(s) is/are correlated to a need for renal replacement therapy.
  • each of the measured concentration(s) may be compared to a threshold value.
  • an increased likelihood of the occurrence of an injury creating a need for renal replacement therapy is assigned to the subject when the measured concentration is above the threshold (relative to the likelihood assigned when the measured concentration is below the threshold); alternatively, when the measured concentration is below the threshold, an increased likelihood of the nonoccurrence of an injury creating a need for renal replacement therapy may be assigned to the subject (relative to the likelihood assigned when the measured concentration is above the threshold).
  • an increased likelihood of the occurrence of an injury creating a need for renal replacement therapy is assigned to the subject when the measured concentration is below the threshold (relative to the likelihood assigned when the measured concentration is above the threshold); alternatively, when the measured concentration is above the threshold, an increased likelihood of the nonoccurrence of an injury creating a need for renal replacement therapy may be assigned to the subject (relative to the likelihood assigned when the measured concentration is below the threshold).
  • these methods comprise diagnosing a subject as being in need of renal transplantation, and the assay result(s ⁇ is/are correlated to a need for renal transplantation.
  • each of the measured concentration(s) may be compared to a threshold value.
  • an increased likelihood of the occurrence of an injury creating a need for renal transplantation is assigned to the subject when the measured concentration is above the threshold (relative to the likelihood assigned when the measured concentration is below the threshold); alternatively, when the measured concentration is below the threshold, an increased likelihood of the nonoccurrence of an injury creating a need for renal transplantation may be assigned to the subject (relative to the likelihood assigned when the measured concentration is above the threshold).
  • an increased likelihood of the occurrence of an injury creating a need for renal transplantation is assigned to the subject when the measured concentration is below the threshold (relative to the likelihood assigned when the measured concentration is above the threshold); alternatively, when the measured concentration is above the threshold, an increased likelihood of the nonoccurrence of an injury creating a need for renal transplantation may be assigned to the subject (relative to the likelihood assigned when the measured concentration is below the threshold).
  • the methods for evaluating renal status described herein are methods for monitoring a renal injury in the subject; that is, assessing whether or not renal function is improving or worsening in a subject who has suffered from an injury to renal function, reduced renal function, or ARF.
  • the assay result(s) for example a measured concentration of one or more markers selected from the group consisting of soluble p-selectin, protein NOV homolog, soluble epidermal growth factor receptor, netrin-4, haptoglobin, heat shock protein beta-1, alpha- 1 -antitrypsin, leukocyte elastase, soluble tumor necrosis factor receptor superfamily member 6, soluble tumor necrosis factor ligand superfamily member 6, soluble intercellular adhesion molecule 2, caspase-3 (and most preferably active caspase-3), and soluble platelet endothelial cell adhesion molecule is/are correlated to the occurrence or nonoccurrence of a change in renal status.
  • the following are preferred monitoring embodiments.
  • these methods comprise monitoring renal status in a subject suffering from an injury to renal function, and the assay result(s) is/are correlated to the occurrence or nonoccurrence of a change in renal status in the subject.
  • the measured concentration(s) may be compared to a threshold value.
  • a threshold value For a positive going marker, when the measured concentration is above the threshold, a worsening of renal function may be assigned to the subject; alternatively, when the measured concentration is below the threshold, an improvement of renal function may be assigned to the subject.
  • a negative going marker when the measured concentration is below the threshold, a worsening of renal function may be assigned to the subject; alternatively, when the measured concentration is above the threshold, an improvement of renal function may be assigned to the subject.
  • these methods comprise monitoring renal status in a subject suffering from reduced renal function, and the assay result(s) is/are correlated to the occurrence or nonoccurrence of a change in renal status in the subject.
  • the measured concentration(s) may be compared to a threshold value.
  • a threshold value For a positive going marker, when the measured concentration is above the threshold, a worsening of renal function may be assigned to the subject; alternatively, when the measured concentration is below the threshold, an improvement of renal function may be assigned to the subject.
  • a negative going marker when the measured concentration is below the threshold, a worsening of renal function may be assigned to the subject; alternatively, when the measured concentration is above the threshold, an improvement of renal function may be assigned to the subject.
  • these methods comprise monitoring renal status in a subject suffering from acute renal failure, and the assay result(s) is/are correlated to the occurrence or nonoccurrence of a change in renal status in the subject.
  • the measured concentration(s) may be compared to a threshold value.
  • a threshold value For a positive going marker, when the measured concentration is above the threshold, a worsening of renal function may be assigned to the subject; alternatively, when the measured concentration is below the threshold, an improvement of renal function may be assigned to the subject.
  • a negative going marker when the measured concentration is below the threshold, a worsening of renal function may be assigned to the subject; alternatively, when the measured concentration is above the threshold, an improvement of renal function may be assigned to the subject.
  • these methods comprise monitoring renal status in a subject at risk of an injury to renal function due to the pre- existence of one or more known risk factors for prerenal, intrinsic renal, or postrenal ARF, and the assay result(s) is/are correlated to the occurrence or nonoccurrence of a change in renal status in the subject.
  • the measured concentration(s) may be compared to a threshold value.
  • a threshold value For a positive going marker, when the measured concentration is above the threshold, a worsening of renal function may be assigned to the subject; alternatively, when the measured concentration is below the threshold, an improvement of renal function may be assigned to the subject.
  • a negative going marker when the measured concentration is below the threshold, a worsening of renal function may be assigned to the subject; alternatively, when the measured concentration is above the threshold, an improvement of renal function may be assigned to the subject.
  • the methods for evaluating renal status described herein are methods for classifying a renal injury in the subject; that is, determining whether a renal injury in a subject is prerenal, intrinsic renal, or postrenal; and/or further subdividing these classes into subclasses such as acute tubular injury, acute glomerulonephritis acute tubulointerstitial nephritis, acute vascular nephropathy, or infiltrative disease; and/or assigning a likelihood that a subject will progress to a particular RIFLE stage.
  • the assay result(s) for example a measured concentration of one or more markers selected from the group consisting of soluble p-selectin, protein NOV homolog, soluble epidermal growth factor receptor, netrin-4, haptoglobin, heat shock protein beta-1, alpha- 1 -antitrypsin, leukocyte elastase, soluble tumor necrosis factor receptor superfamily member 6, soluble tumor necrosis factor ligand superfamily member 6, soluble intercellular adhesion molecule 2, caspase-3 (and most preferably active caspase-3), and soluble platelet endothelial cell adhesion molecule is/are correlated to a particular class and/or subclass.
  • these methods comprise determining whether a renal injury in a subject is prerenal, intrinsic renal, or postrenal; and/or further subdividing these classes into subclasses such as acute tubular injury, acute glomerulonephritis acute tubulointerstitial nephritis, acute vascular nephropathy, or infiltrative disease; and/or assigning a likelihood that a subject will progress to a particular RIFLE stage, and the assay result(s) is/are correlated to the injury classification for the subject. For example, the measured concentration may be compared to a threshold value, and when the measured concentration is above the threshold, a particular classification is assigned; alternatively, when the measured concentration is below the threshold, a different classification may be assigned to the subject.
  • the threshold value may be determined from a population of normal subjects by selecting a concentration representing the 75 th , 85 th , 90 th , 95 th , or 99 th percentile of a kidney injury marker measured in such normal subjects.
  • the threshold value may be determined from a "diseased" population of subjects, e.g., those suffering from an injury or having a predisposition for an injury (e.g., progression to ARF or some other clinical outcome such as death, dialysis, renal transplantation, etc.), by selecting a concentration representing the 75 th , 85 th , 90 th , 95 th , or 99 th percentile of a kidney injury marker measured in such subjects.
  • the threshold value may be determined from a prior measurement of a kidney injury marker in the same subject; that is, a temporal change in the level of a kidney injury marker in the subject may be used to assign risk to the subject.
  • kidney injury markers of the present invention must be compared to corresponding individual thresholds.
  • Methods for combining assay results can comprise the use of multivariate logistical regression, loglinear modeling, neural network analysis, n-of-m analysis, decision tree analysis, calculating ratios of markers, etc. This list is not meant to be limiting.
  • a composite result which is determined by combining individual markers may be treated as if it is itself a marker; that is, a threshold may be determined for the composite result as described herein for individual markers, and the composite result for an individual patient compared to this threshold.
  • ROC curves established from a "first" subpopulation which is predisposed to one or more future changes in renal status, and a "second" subpopulation which is not so predisposed can be used to calculate a ROC curve, and the area under the curve provides a measure of the quality of the test.
  • the tests described herein provide a ROC curve area greater than 0.5, preferably at least 0.6, more preferably 0.7, still more preferably at least 0.8, even more preferably at least 0.9, and most preferably at least 0.95.
  • the measured concentration of one or more kidney injury markers, or a composite of such markers may be treated as continuous variables.
  • any particular concentration can be converted into a corresponding probability of a future reduction in renal function for the subject, the occurrence of an injury, a classification, etc.
  • a threshold that can provide an acceptable level of specificity and sensitivity in separating a population of subjects into "bins” such as a "first" subpopulation (e.g., which is predisposed to one or more future changes in renal status, the occurrence of an injury, a classification, etc.) and a "second" subpopulation which is not so predisposed.
  • a threshold value is selected to separate this first and second population by one or more of the following measures of test accuracy: an odds ratio greater than 1, preferably at least about 2 or more or about 0.5 or less, more preferably at least about 3 or more or about 0.33 or less, still more preferably at least about 4 or more or about 0.25 or less, even more preferably at least about 5 or more or about 0.2 or less, and most preferably at least about 10 or more or about 0.1 or less; a specificity of greater than 0.5, preferably at least about 0.6, more preferably at least about 0.7, still more preferably at least about 0.8, even more preferably at least about 0.9 and most preferably at least about 0.95, with a corresponding sensitivity greater than 0.2, preferably greater than about 0.3, more preferably greater than about 0.4, still more preferably at least about 0.5, even more preferably about 0.6, yet more preferably greater than about 0.7, still more preferably greater than about 0.8, more preferably greater than about 0.9, and most preferably greater than about 0.95;
  • Multiple thresholds may also be used to assess renal status in a subject. For example, a "first" subpopulation which is predisposed to one or more future changes in renal status, the occurrence of an injury, a classification, etc., and a "second" subpopulation which is not so predisposed can be combined into a single group. This group is then subdivided into three or more equal parts (known as tertiles, quartiles, quintiles, etc., depending on the number of subdivisions). An odds ratio is assigned to subjects based on which subdivision they fall into. If one considers a tertile, the lowest or highest tertile can be used as a reference for comparison of the other subdivisions. This reference subdivision is assigned an odds ratio of 1.
  • the second tertile is assigned an odds ratio that is relative to that first tertile. That is, someone in the second tertile might be 3 times more likely to suffer one or more future changes in renal status in comparison to someone in the first tertile.
  • the third tertile is also assigned an odds ratio that is relative to that first tertile.
  • the assay method is an immunoassay.
  • Antibodies for use in such assays will specifically bind a full length kidney injury marker of interest, and may also bind one or more polypeptides that are "related" thereto, as that term is defined hereinafter. Numerous immunoassay formats are known to those of skill in the art.
  • Preferred body fluid samples are selected from the group consisting of urine, blood, serum, saliva, tears, and plasma.
  • kidney injury marker assay result(s) is/are used in isolation in the methods described herein. Rather, additional variables or other clinical indicia may be included in the methods described herein. For example, a risk stratification, diagnostic, classification, monitoring, etc.
  • method may combine the assay result(s) with one or more variables measured for the subject selected from the group consisting of demographic information (e.g., weight, sex, age, race), medical history (e.g., family history, type of surgery, pre-existing disease such as aneurism, congestive heart failure, preeclampsia, eclampsia, diabetes mellitus, hypertension, coronary artery disease, proteinuria, renal insufficiency, or sepsis, type of toxin exposure such as NSAIDs, cyclosporines, tacrolimus, aminoglycosides, foscarnet, ethylene glycol, hemoglobin, myoglobin, ifosfamide, heavy metals, methotrexate, radiopaque contrast agents, or strep tozotocin), clinical variables (e.g., blood pressure, temperature, respiration rate), risk scores (APACHE score, PREDICT score, TIMI Risk Score for UA/NSTEMI, Fra
  • kidney injury marker assay result(s) Other measures of renal function which may be combined with one or more kidney injury marker assay result(s) are described hereinafter and in Harrison's Principles of Internal Medicine, 17 th Ed., McGraw Hill, New York, pages 1741-1830, and Current Medical Diagnosis & Treatment 2008, 47 th Ed, McGraw Hill, New York, pages 785-815, each of which are hereby incorporated by reference in their entirety.
  • the individual markers may be measured in samples obtained at the same time, or may be determined from samples obtained at different (e.g., an earlier or later) times.
  • the individual markers may also be measured on the same or different body fluid samples. For example, one kidney injury marker may be measured in a serum or plasma sample and another kidney injury marker may be measured in a urine sample.
  • assignment of a likelihood may combine an individual kidney injury marker assay result with temporal changes in one or more additional variables.
  • kits for performing the methods described herein comprise reagents sufficient for performing an assay for at least one of the described kidney injury markers, together with instructions for performing the described threshold comparisons.
  • reagents for performing such assays are provided in an assay device, and such assay devices may be included in such a kit.
  • Preferred reagents can comprise one or more solid phase antibodies, the solid phase antibody comprising antibody that detects the intended biomarker target(s) bound to a solid support.
  • such reagents can also include one or more detectably labeled antibodies, the detectably labeled antibody comprising antibody that detects the intended biomarker target(s) bound to a detectable label. Additional optional elements that may be provided as part of an assay device are described hereinafter.
  • Detectable labels may include molecules that are themselves detectable (e.g., fluorescent moieties, electrochemical labels, eel (electrochemical luminescence) labels, metal chelates, colloidal metal particles, etc.) as well as molecules that may be indirectly detected by production of a detectable reaction product (e.g., enzymes such as horseradish peroxidase, alkaline phosphatase, etc.) or through the use of a specific binding molecule which itself may be detectable (e.g., a labeled antibody that binds to the second antibody, biotin, digoxigenin, maltose, oligohistidine, 2,4-dintrobenzene, phenylarsenate, ssDNA, dsDNA, etc.).
  • a detectable reaction product e.g., enzymes such as horseradish peroxidase, alkaline phosphatase, etc.
  • a specific binding molecule which itself may be detectable (e.g.,
  • Generation of a signal from the signal development element can be performed using various optical, acoustical, and electrochemical methods well known in the art.
  • detection modes include fluorescence, radiochemical detection, reflectance, absorbance, amperometry, conductance, impedance, interferometry, ellipsometry, etc.
  • the solid phase antibody is coupled to a transducer (e.g., a diffraction grating, electrochemical sensor, etc) for generation of a signal, while in others, a signal is generated by a transducer that is spatially separate from the solid phase antibody (e.g., a fluorometer that employs an excitation light source and an optical detector).
  • a transducer e.g., a diffraction grating, electrochemical sensor, etc
  • a signal is generated by a transducer that is spatially separate from the solid phase antibody (e.g., a fluorometer that employs an excitation light source and an optical detector).
  • the present invention relates to methods and compositions for diagnosis, differential diagnosis, risk stratification, monitoring, classifying and determination of treatment regimens in subjects suffering or at risk of suffering from injury to renal function, reduced renal function and/or acute renal failure through measurement of one or more kidney injury markers.
  • an "injury to renal function” is an abrupt (within 14 days, preferably within 7 days, more preferably within 72 hours, and still more preferably within 48 hours) measurable reduction in a measure of renal function. Such an injury may be identified, for example, by a decrease in glomerular filtration rate or estimated GFR, a reduction in urine output, an increase in serum creatinine, an increase in serum cystatin C, a requirement for renal replacement therapy, etc.
  • "Improvement in Renal Function” is an abrupt (within 14 days, preferably within 7 days, more preferably within 72 hours, and still more preferably within 48 hours) measurable increase in a measure of renal function. Preferred methods for measuring and/or estimating GFR are described hereinafter.
  • reduced renal function is an abrupt (within 14 days, preferably within 7 days, more preferably within 72 hours, and still more preferably within 48 hours) reduction in kidney function identified by an absolute increase in serum creatinine of greater than or equal to 0.1 mg/dL (> 8.8 ⁇ mol/L), a percentage increase in serum creatinine of greater than or equal to 20% (1.2-fold from baseline), or a reduction in urine output (documented oliguria of less than 0. 5 ml/kg per hour).
  • Acute renal failure is an abrupt (within 14 days, preferably within 7 days, more preferably within 72 hours, and still more preferably within 48 hours) reduction in kidney function identified by an absolute increase in serum creatinine of greater than or equal to 0.3 mg/dl (> 26.4 ⁇ mol/1), a percentage increase in serum creatinine of greater than or equal to 50% (1. 5-fold from baseline), or a reduction in urine output (documented oliguria of less than 0.5 ml/kg per hour for at least 6 hours).
  • This term is synonymous with "acute kidney injury" or "AKI.”
  • the signals obtained from an immunoassay are a direct result of complexes formed between one or more antibodies and the target biomolecule (i.e., the analyte) and polypeptides containing the necessary epitope(s) to which the antibodies bind. While such assays may detect the full length biomarker and the assay result be expressed as a concentration of a biomarker of interest, the signal from the assay is actually a result of all such "immunoreactive" polypeptides present in the sample.
  • Biomarkers may also be determined by means other than immunoassays, including protein measurements (such as dot blots, western blots, chromatographic methods, mass spectrometry, etc.) and nucleic acid measurements (mRNA quatitation). This list is not meant to be limiting.
  • p-selectin refers to one or more polypeptides present in a biological sample that are derived from the p-selectin precursor (Swiss-Prot P16109 (SEQ ID NO: I)).
  • the p-selectin assay detects one or more soluble forms of p- selectin.
  • P-selectin is a single-pass type I membrane protein having a large extracellular domain, most or all of which is present in soluble forms of p-selectin generated either through alternative splicing event which deletes all or a portion of the transmembrane domain, or by proteolysis of the membrane-bound form.
  • one or more antibodies that bind to epitopes within this extracellular domain may be used to detect these soluble form(s). The following domains have been identified in p-selectin:
  • protein NOV homolog refers to one or more polypeptides present in a biological sample that are derived from the protein NOV homolog precursor (Swiss-Prot P48745 (SEQ ID NO: 2)).
  • LGGLTLAAYR PEATLGVEVS DSSVNCIEQT TEWTACSKSC GMGFSTRVTN RNRQCEMLKQ
  • epidermal growth factor receptor refers to one or more polypeptides present in a biological sample that are derived from the epidermal growth factor receptor precursor (Swiss-Prot P00533 (SEQ ID NO: 3)).
  • SIDDTFLPVP EYINQSVPKR PAGSVQNPVY HNQPLNPAPS RDPHYQDPHS TAVGNPEYLN
  • the epidermal growth factor receptor assay detects one or more soluble forms of epidermal growth factor receptor.
  • Epidermal growth factor receptor is a single-pass type I membrane protein having a large extracellular domain, most or all of which is present in soluble forms of epidermal growth factor receptor generated either through alternative splicing event which deletes all or a portion of the transmembrane domain, or by proteolysis of the membrane-bound form.
  • an immunoassay one or more antibodies that bind to epitopes within this extracellular domain may be used to detect these soluble form(s). The following domains have been identified in epidermal growth factor receptor:
  • netrin-4" refers to one or more polypeptides present in a biological sample that are derived from the netrin-4 precursor (Swiss-Prot Q9HB63 (SEQ ID NO: 4)).
  • haptoglobin refers to one or more polypeptides present in a biological sample that are derived from the haptoglobin precursor (Swiss-Prot P00738 (SEQ ID NO: 5)).
  • haptoglobin [0060] The following domains have been identified in haptoglobin:
  • alpha- 1 -antitrypsin refers to one or more polypeptides present in a biological sample that are derived from the alpha- 1- antitrypsin precursor (Swiss- Prot P01009 (SEQ ID NO: 6)).
  • leukocyte elastase refers to one or more polypeptides present in a biological sample that are derived from the leukocyte elastase precursor (Swiss- Prot P08246 (SEQ ID NO: 7)).
  • tumor necrosis factor receptor superfamily member 6 refers to one or more polypeptides present in a biological sample that are derived from the tumor necrosis factor receptor superfamily member 6 precursor (Swiss-Prot P25445 (SEQ ID NO: 8)).
  • the tumor necrosis factor receptor superfamily member 6 assay detects one or more soluble forms of tumor necrosis factor receptor superfamily member 6.
  • Tumor necrosis factor receptor superfamily member 6 is a single-pass type I membrane protein having a large extracellular domain, most or all of which is present in soluble forms of tumor necrosis factor receptor superfamily member 6 generated either through alternative splicing event which deletes all or a portion of the transmembrane domain, or by proteolysis of the membrane-bound form.
  • an immunoassay one or more antibodies that bind to epitopes within this extracellular domain may be used to detect these soluble form(s). The following domains have been identified in tumor necrosis factor receptor superfamily member 6:
  • tumor necrosis factor ligand superfamily member 6 refers to one or more polypeptides present in a biological sample that are derived from the tumor necrosis factor ligand superfamily member 6 precursor (Swiss-Prot P48023 (SEQ ID NO: 9)). 10 20 30 40 50 60
  • the tumor necrosis factor ligand superfamily member 6 assay detects one or more soluble forms of tumor necrosis factor ligand superfamily member 6.
  • Tumor necrosis factor ligand superfamily member 6 is a single-pass type II membrane protein having a large extracellular domain, most or all of which is present in soluble forms of tumor necrosis factor ligand superfamily member 6 generated either through alternative splicing event which deletes all or a portion of the transmembrane domain, or by proteolysis of the membrane-bound form.
  • an immunoassay one or more antibodies that bind to epitopes within this extracellular domain may be used to detect these soluble form(s). The following domains have been identified in tumor necrosis factor ligand superfamily member 6:
  • tumor necrosis factor ligand superfamily member 6 soluble form
  • intercellular adhesion molecule 2 refers to one or more polypeptides present in a biological sample that are derived from the intercellular adhesion molecule 2 precursor (Swiss-Prot P13598 (SEQ ID NO: 10)).
  • the intercellular adhesion molecule 2 assay detects one or more soluble forms of intercellular adhesion molecule 2.
  • Intercellular adhesion molecule 2 is a single-pass type I membrane protein having a large extracellular domain, most or all of which is present in soluble forms of intercellular adhesion molecule 2 generated either through alternative splicing event which deletes all or a portion of the transmembrane domain, or by proteolysis of the membrane-bound form.
  • one or more antibodies that bind to epitopes within this extracellular domain may be used to detect these soluble form(s). The following domains have been identified in intercellular adhesion molecule 2:
  • caspase-3 refers to one or more polypeptides present in a biological sample that are derived from the caspase-3 precursor (Swiss-Prot P42574 (SEQ ID NO: H)).
  • Suitable assays may recognize only the pl7 subunit of caspase-3, may recognize only the pl2 subunit of caspase-3 (24 kDa) but not the full length caspase-3, may recognize only full length caspase-3, or may recognize one subunit and the full length full length caspase-3.
  • the skilled artisan will understand that the signals obtained from an immunoassay are a direct result of complexes formed between one or more antibodies and the target biomolecule (i.e., the analyte) and polypeptides containing the necessary epitope(s) to which the antibodies bind.
  • platelet endothelial cell adhesion molecule refers to one or more polypeptides present in a biological sample that are derived from the platelet endothelial cell adhesion molecule precursor (Swiss-Prot P16284 (SEQ ID NO: 12)).
  • the platelet endothelial cell adhesion molecule assay detects one or more soluble forms of platelet endothelial cell adhesion molecule.
  • Platelet endothelial cell adhesion molecule is a single-pass type I membrane protein having a large extracellular domain, most or all of which is present in soluble forms of platelet endothelial cell adhesion molecule generated either through alternative splicing event which deletes all or a portion of the transmembrane domain, or by proteolysis of the membrane-bound form.
  • an immunoassay one or more antibodies that bind to epitopes within this extracellular domain may be used to detect these soluble form(s). The following domains have been identified in platelet endothelial cell adhesion molecule:
  • heat shock protein beta-1 refers to one or more polypeptides present in a biological sample that are derived from the heat shock protein beta- 1 precursor (Swiss-Prot P04792 (SEQ ID NO: 13)).
  • epidermal growth factor receptor refers to one or more polypeptides present in a biological sample that are derived from the epidermal growth factor receptor precursor (Swiss-Prot P00533 (SEQ ID NO: 14)). 10 20 30 40 50 60
  • SIDDTFLPVP EYINQSVPKR PAGSVQNPVY HNQPLNPAPS RDPHYQDPHS TAVGNPEYLN
  • the epidermal growth factor receptor assay detects one or more soluble forms of epidermal growth factor receptor.
  • Epidermal growth factor receptor is a single-pass type I membrane protein having a large extracellular domain, most or all of which is present in soluble forms of epidermal growth factor receptor generated either through alternative splicing event which deletes all or a portion of the transmembrane domain, or by proteolysis of the membrane-bound form.
  • an immunoassay one or more antibodies that bind to epitopes within this extracellular domain may be used to detect these soluble form(s). The following domains have been identified in epidermal growth factor receptor:
  • the term "relating a signal to the presence or amount" of an analyte reflects this understanding. Assay signals are typically related to the presence or amount of an analyte through the use of a standard curve calculated using known concentrations of the analyte of interest. As the term is used herein, an assay is "configured to detect" an analyte if an assay can generate a detectable signal indicative of the presence or amount of a physiologically relevant concentration of the analyte.
  • an immunoassay configured to detect a marker of interest will also detect polypeptides related to the marker sequence, so long as those polypeptides contain the epitope(s) necessary to bind to the antibody or antibodies used in the assay.
  • the term "related marker” as used herein with regard to a biomarker such as one of the kidney injury markers described herein refers to one or more fragments, variants, etc., of a particular marker or its biosynthetic parent that may be detected as a surrogate for the marker itself or as independent biomarkers.
  • the term also refers to one or more polypeptides present in a biological sample that are derived from the biomarker precursor complexed to additional species, such as binding proteins, receptors, heparin, lipids, sugars, etc.
  • positive going marker refers to a marker that is determined to be elevated in subjects suffering from a disease or condition, relative to subjects not suffering from that disease or condition.
  • negative going marker refers to a marker that is determined to be reduced in subjects suffering from a disease or condition, relative to subjects not suffering from that disease or condition.
  • subject refers to a human or non-human organism.
  • methods and compositions described herein are applicable to both human and veterinary disease.
  • a subject is preferably a living organism, the invention described herein may be used in post-mortem analysis as well.
  • Preferred subjects are humans, and most preferably "patients,” which as used herein refers to living humans that are receiving medical care for a disease or condition. This includes persons with no defined illness who are being investigated for signs of pathology.
  • an analyte is measured in a sample.
  • a sample may be obtained from a subject, or may be obtained from biological materials intended to be provided to the subject.
  • a sample may be obtained from a kidney being evaluated for possible transplantation into a subject, and an analyte measurement used to evaluate the kidney for preexisting damage.
  • Preferred samples are body fluid samples.
  • body fluid sample refers to a sample of bodily fluid obtained for the purpose of diagnosis, prognosis, classification or evaluation of a subject of interest, such as a patient or transplant donor.
  • such a sample may be obtained for the purpose of determining the outcome of an ongoing condition or the effect of a treatment regimen on a condition.
  • Preferred body fluid samples include blood, serum, plasma, cerebrospinal fluid, urine, saliva, sputum, and pleural effusions.
  • body fluid samples would be more readily analyzed following a fractionation or purification procedure, for example, separation of whole blood into serum or plasma components.
  • diagnosis refers to methods by which the skilled artisan can estimate and/or determine the probability ("a likelihood") of whether or not a patient is suffering from a given disease or condition.
  • diagnosis includes using the results of an assay, most preferably an immunoassay, for a kidney injury marker of the present invention, optionally together with other clinical characteristics, to arrive at a diagnosis (that is, the occurrence or nonoccurrence) of an acute renal injury or ARF for the subject from which a sample was obtained and assayed. That such a diagnosis is "determined” is not meant to imply that the diagnosis is 100% accurate. Many biomarkers are indicative of multiple conditions.
  • a measured biomarker level on one side of a predetermined diagnostic threshold indicates a greater likelihood of the occurrence of disease in the subject relative to a measured level on the other side of the predetermined diagnostic threshold.
  • a prognostic risk signals a probability ("a likelihood") that a given course or outcome will occur.
  • a level or a change in level of a prognostic indicator which in turn is associated with an increased probability of morbidity (e.g., worsening renal function, future ARF, or death) is referred to as being "indicative of an increased likelihood" of an adverse outcome in a patient.
  • immunoassays involve contacting a sample containing or suspected of containing a biomarker of interest with at least one antibody that specifically binds to the biomarker. A signal is then generated indicative of the presence or amount of complexes formed by the binding of polypeptides in the sample to the antibody. The signal is then related to the presence or amount of the biomarker in the sample. Numerous methods and devices are well known to the skilled artisan for the detection and analysis of biomarkers. See, e.g., U.S.
  • the assay devices and methods known in the art can utilize labeled molecules in various sandwich, competitive, or non-competitive assay formats, to generate a signal that is related to the presence or amount of the biomarker of interest.
  • Suitable assay formats also include chromatographic, mass spectrographic, and protein "blotting" methods.
  • certain methods and devices such as biosensors and optical immunoassays, may be employed to determine the presence or amount of analytes without the need for a labeled molecule. See, e.g., U.S. Patents 5,631,171; and 5,955,377, each of which is hereby incorporated by reference in its entirety, including all tables, figures and claims.
  • robotic instrumentation including but not limited to Beckman ACCESS®, Abbott AXSYM®, Roche ELECSYS®, Dade Behring STRATUS® systems are among the immunoassay analyzers that are capable of performing immunoassays.
  • any suitable immunoassay may be utilized, for example, enzyme-linked immunoassays (ELISA), radioimmunoassays (RIAs), competitive binding assays, and the like.
  • Antibodies or other polypeptides may be immobilized onto a variety of solid supports for use in assays.
  • Solid phases that may be used to immobilize specific binding members include include those developed and/or used as solid phases in solid phase binding assays. Examples of suitable solid phases include membrane filters, cellulose-based papers, beads (including polymeric, latex and paramagnetic particles), glass, silicon wafers, microparticles, nanoparticles, TentaGels, AgroGels, PEGA gels, SPOCC gels, and multiple- well plates.
  • An assay strip could be prepared by coating the antibody or a plurality of antibodies in an array on solid support.
  • Antibodies or other polypeptides may be bound to specific zones of assay devices either by conjugating directly to an assay device surface, or by indirect binding. In an example of the later case, antibodies or other polypeptides may be immobilized on particles or other solid supports, and that solid support immobilized to the device surface.
  • Biological assays require methods for detection, and one of the most common methods for quantitation of results is to conjugate a detectable label to a protein or nucleic acid that has affinity for one of the components in the biological system being studied.
  • Detectable labels may include molecules that are themselves detectable (e.g., fluorescent moieties, electrochemical labels, metal chelates, etc.) as well as molecules that may be indirectly detected by production of a detectable reaction product (e.g., enzymes such as horseradish peroxidase, alkaline phosphatase, etc.) or by a specific binding molecule which itself may be detectable (e.g., biotin, digoxigenin, maltose, oligohistidine, 2,4-dintrobenzene, phenylarsenate, ssDNA, dsDNA, etc.).
  • a detectable reaction product e.g., enzymes such as horseradish peroxidase, alkaline phosphatase, etc.
  • Cross-linking reagents contain at least two reactive groups, and are divided generally into homofunctional cross-linkers (containing identical reactive groups) and heterofunctional cross-linkers (containing non-identical reactive groups). Homobifunctional cross-linkers that couple through amines, sulfhydryls or react non- specifically are available from many commercial sources. Maleimides, alkyl and aryl halides, alpha-haloacyls and pyridyl disulfides are thiol reactive groups.
  • kits for the analysis of the described kidney injury markers comprises reagents for the analysis of at least one test sample which comprise at least one antibody that a kidney injury marker.
  • the kit can also include devices and instructions for performing one or more of the diagnostic and/or prognostic correlations described herein.
  • Preferred kits will comprise an antibody pair for performing a sandwich assay, or a labeled species for performing a competitive assay, for the analyte.
  • an antibody pair comprises a first antibody conjugated to a solid phase and a second antibody conjugated to a detectable label, wherein each of the first and second antibodies that bind a kidney injury marker.
  • each of the antibodies are monoclonal antibodies.
  • the instructions for use of the kit and performing the correlations can be in the form of labeling, which refers to any written or recorded material that is attached to, or otherwise accompanies a kit at any time during its manufacture, transport, sale or use.
  • labeling encompasses advertising leaflets and brochures, packaging materials, instructions, audio or video cassettes, computer discs, as well as writing imprinted directly on kits.
  • antibody refers to a peptide or polypeptide derived from, modeled after or substantially encoded by an immunoglobulin gene or immunoglobulin genes, or fragments thereof, capable of specifically binding an antigen or epitope. See, e.g. Fundamental Immunology, 3rd Edition, W.E. Paul, ed., Raven Press, N.Y. (1993); Wilson (1994; J. Immunol. Methods 175:267-273; Yarmush (1992) J. Biochem. Biophys. Methods 25:85-97.
  • antibody includes antigen-binding portions, i.e., "antigen binding sites,” (e.g., fragments, subsequences, complementarity determining regions (CDRs)) that retain capacity to bind antigen, including (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CHl domains; (ii) a F(ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VH and CHl domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (v) a dAb fragment (Ward et al., (1989) Nature 341:544-546), which consists of a VH domain; and (vi) an isolated complementarity determining region (CDR).
  • Antigen binding sites e.g., fragments, subs
  • Antibodies used in the immunoassays described herein preferably specifically bind to a kidney injury marker of the present invention.
  • the term “specifically binds” is not intended to indicate that an antibody binds exclusively to its intended target since, as noted above, an antibody binds to any polypeptide displaying the epitope(s) to which the antibody binds. Rather, an antibody "specifically binds” if its affinity for its intended target is about 5- fold greater when compared to its affinity for a non-target molecule which does not display the appropriate epitope(s).
  • the affinity of the antibody will be at least about 5 fold, preferably 10 fold, more preferably 25-fold, even more preferably 50-fold, and most preferably 100-fold or more, greater for a target molecule than its affinity for a non-target molecule.
  • Preferred antibodies bind with affinities of at least about 10 7 M "1 , and preferably between about 10 8 M “1 to about 10 9 M “1 , about 10 9 M “1 to about 10 10 M “1 , or about 10 10 M “1 to about 10 12 M "1 .
  • Affinity can be determined at equilibrium by measuring the fraction bound (r) of labeled ligand at various concentrations (c).
  • r/c is plotted on the Y-axis versus r on the X- axis, thus producing a Scatchard plot.
  • Antibody affinity measurement by Scatchard analysis is well known in the art. See, e.g., van Erp et al, J. Immunoassay 12: 425-43, 1991; Nelson and Griswold, Comput. Methods Programs Biomed. 27: 65-8, 1988.
  • epitope refers to an antigenic determinant capable of specific binding to an antibody.
  • Epitopes usually consist of chemically active surface groupings of molecules such as amino acids or sugar side chains and usually have specific three dimensional structural characteristics, as well as specific charge characteristics. Conformational and nonconformational epitopes are distinguished in that the binding to the former but not the latter is lost in the presence of denaturing solvents.
  • phage display technology to produce and screen libraries of polypeptides for binding to a selected analyte. See, e.g, Cwirla et al., Proc. Natl. Acad. ScL USA 87, 6378-82, 1990; Devlin et al, Science 249, 404-6, 1990, Scott and Smith, Science 249, 386-88, 1990; and Ladner et al, U.S. Pat. No. 5,571,698.
  • a basic concept of phage display methods is the establishment of a physical association between DNA encoding a polypeptide to be screened and the polypeptide.
  • This physical association is provided by the phage particle, which displays a polypeptide as part of a capsid enclosing the phage genome which encodes the polypeptide.
  • the establishment of a physical association between polypeptides and their genetic material allows simultaneous mass screening of very large numbers of phage bearing different polypeptides.
  • Phage displaying a polypeptide with affinity to a target bind to the target and these phage are enriched by affinity screening to the target.
  • the identity of polypeptides displayed from these phage can be determined from their respective genomes. Using these methods a polypeptide identified as having a binding affinity for a desired target can then be synthesized in bulk by conventional means. See, e.g., U.S. Patent No.
  • the antibodies that are generated by these methods may then be selected by first screening for affinity and specificity with the purified polypeptide of interest and, if required, comparing the results to the affinity and specificity of the antibodies with polypeptides that are desired to be excluded from binding.
  • the screening procedure can involve immobilization of the purified polypeptides in separate wells of microtiter plates. The solution containing a potential antibody or groups of antibodies is then placed into the respective microtiter wells and incubated for about 30 min to 2 h.
  • microtiter wells are then washed and a labeled secondary antibody (for example, an anti-mouse antibody conjugated to alkaline phosphatase if the raised antibodies are mouse antibodies) is added to the wells and incubated for about 30 min and then washed. Substrate is added to the wells and a color reaction will appear where antibody to the immobilized polypeptide(s) are present.
  • a labeled secondary antibody for example, an anti-mouse antibody conjugated to alkaline phosphatase if the raised antibodies are mouse antibodies
  • the antibodies so identified may then be further analyzed for affinity and specificity in the assay design selected.
  • the purified target protein acts as a standard with which to judge the sensitivity and specificity of the immunoassay using the antibodies that have been selected. Because the binding affinity of various antibodies may differ; certain antibody pairs (e.g., in sandwich assays) may interfere with one another sterically, etc., assay performance of an antibody may be a more important measure than absolute affinity and specificity of an antibody.
  • correlating refers to comparing the presence or amount of the biomarker(s) in a patient to its presence or amount in persons known to suffer from, or known to be at risk of, a given condition; or in persons known to be free of a given condition. Often, this takes the form of comparing an assay result in the form of a biomarker concentration to a predetermined threshold selected to be indicative of the occurrence or nonoccurrence of a disease or the likelihood of some future outcome.
  • Selecting a diagnostic threshold involves, among other things, consideration of the probability of disease, distribution of true and false diagnoses at different test thresholds, and estimates of the consequences of treatment (or a failure to treat) based on the diagnosis. For example, when considering administering a specific therapy which is highly efficacious and has a low level of risk, few tests are needed because clinicians can accept substantial diagnostic uncertainty. On the other hand, in situations where treatment options are less effective and more risky, clinicians often need a higher degree of diagnostic certainty. Thus, cost/benefit analysis is involved in selecting a diagnostic threshold.
  • Suitable thresholds may be determined in a variety of ways. For example, one recommended diagnostic threshold for the diagnosis of acute myocardial infarction using cardiac troponin is the 97.5 th percentile of the concentration seen in a normal population. Another method may be to look at serial samples from the same patient, where a prior "baseline" result is used to monitor for temporal changes in a biomarker level.
  • ROC Reciever Operating Characteristic
  • the ROC graph is sometimes called the sensitivity vs (1 - specificity) plot.
  • a perfect test will have an area under the ROC curve of 1.0; a random test will have an area of 0.5.
  • a threshold is selected to provide an acceptable level of specificity and sensitivity.
  • diseased is meant to refer to a population having one characteristic (the presence of a disease or condition or the occurrence of some outcome) and “nondiseased” is meant to refer to a population lacking the characteristic. While a single decision threshold is the simplest application of such a method, multiple decision thresholds may be used. For example, below a first threshold, the absence of disease may be assigned with relatively high confidence, and above a second threshold the presence of disease may also be assigned with relatively high confidence. Between the two thresholds may be considered indeterminate. This is meant to be exemplary in nature only.
  • Measures of test accuracy may be obtained as described in Fischer et al, Intensive Care Med. 29: 1043-51, 2003, and used to determine the effectiveness of a given biomarker. These measures include sensitivity and specificity, predictive values, likelihood ratios, diagnostic odds ratios, and ROC curve areas.
  • the area under the curve ("AUC") of a ROC plot is equal to the probability that a classifier will rank a randomly chosen positive instance higher than a randomly chosen negative one.
  • the area under the ROC curve may be thought of as equivalent to the Mann-Whitney U test, which tests for the median difference between scores obtained in the two groups considered if the groups are of continuous data, or to the Wilcoxon test of ranks.
  • suitable tests may exhibit one or more of the following results on these various measures: a specificity of greater than 0.5, preferably at least 0.6, more preferably at least 0.7, still more preferably at least 0.8, even more preferably at least 0.9 and most preferably at least 0.95, with a corresponding sensitivity greater than 0.2, preferably greater than 0.3, more preferably greater than 0.4, still more preferably at least 0.5, even more preferably 0.6, yet more preferably greater than 0.7, still more preferably greater than 0.8, more preferably greater than 0.9, and most preferably greater than 0.95; a sensitivity of greater than 0.5, preferably at least 0.6, more preferably at least 0.7, still more preferably at least 0.8, even more preferably at least 0.9 and most preferably at least 0.95, with a corresponding specificity greater than 0.2, preferably greater than 0.3, more preferably greater than 0.4, still more preferably at least 0.5, even more preferably 0.6, yet more preferably greater than 0.7
  • Additional clinical indicia may be combined with the kidney injury marker assay result(s) of the present invention.
  • biomarkers related to renal status include the following, which recite the common biomarker name, followed by the Swiss-Prot entry number for that biomarker or its parent: Actin (P68133); Adenosine deaminase binding protein (DPP4, P27487); Alpha- 1-acid glycoprotein 1 (P02763); Alpha- 1 -microglobulin (P02760); Albumin (P02768); Angiotensinogenase (Renin, P00797); Annexin A2 (P07355); Beta-glucuronidase (P08236); B-2-microglobulin (P61679); Beta- galactosidase (P16278); BMP-7 (P18075); Brain natriuretic peptide (proBNP, BNP-32, NTproBNP; P16860); Calcium-binding protein Beta (P68133); Aden
  • Adiponectin (Q15848); Alkaline phosphatase (P05186); Aminopeptidase N (P15144); CalbindinD28k (P05937); Cystatin C (P01034); 8 subunit of FIFO ATPase (P03928); Gamma-glutamyltransferase (P19440); GSTa (alpha- glutathione-S-transferase, P08263); GSTpi (Glutathione-S-transferase P; GST class-pi; P09211); IGFBP-I (P08833); IGFBP-2 (P18065); IGFBP-6 (P24592); Integral membrane protein 1 (Itml, P46977); Interleukin-6 (P05231); Interleukin-8 (P10145); Interleukin-18 (Q14116); IP-IO (10 kDa interferon-gamma-induced protein, P
  • Other clinical indicia which may be combined with the kidney injury marker assay result(s) of the present invention includes demographic information (e.g., weight, sex, age, race), medical history (e.g., family history, type of surgery, pre-existing disease such as aneurism, congestive heart failure, preeclampsia, eclampsia, diabetes mellitus, hypertension, coronary artery disease, proteinuria, renal insufficiency, or sepsis, type of toxin exposure such as NSAIDs, cyclosporines, tacrolimus, aminoglycosides, foscarnet, ethylene glycol, hemoglobin, myoglobin, ifosfamide, heavy metals, methotrexate, radiopaque contrast agents, or strep tozotocin), clinical variables (e.g., blood pressure, temperature, respiration rate), risk scores (APACHE score, PREDICT score, TIMI Risk Score for UA/NSTEM
  • kidney injury marker assay result(s) Other measures of renal function which may be combined with the kidney injury marker assay result(s) are described hereinafter and in Harrison's Principles of Internal Medicine, 17 th Ed., McGraw Hill, New York, pages 1741- 1830, and Current Medical Diagnosis & Treatment 2008, 47 th Ed, McGraw Hill, New York, pages 785-815, each of which are hereby incorporated by reference in their entirety.
  • Combining assay results/clinical indicia in this manner can comprise the use of multivariate logistical regression, loglinear modeling, neural network analysis, n-of-m analysis, decision tree analysis, etc. This list is not meant to be limiting.
  • the terms "acute renal (or kidney) injury” and “acute renal (or kidney) failure” as used herein are defined in part in terms of changes in serum creatinine from a baseline value.
  • Most definitions of ARF have common elements, including the use of serum creatinine and, often, urine output. Patients may present with renal dysfunction without an available baseline measure of renal function for use in this comparison. In such an event, one may estimate a baseline serum creatinine value by assuming the patient initially had a normal GFR.
  • Glomerular filtration rate (GFR) is the volume of fluid filtered from the renal (kidney) glomerular capillaries into the Bowman's capsule per unit time. Glomerular filtration rate (GFR) can be calculated by measuring any chemical that has a steady level in the blood, and is freely filtered but neither reabsorbed nor secreted by the kidneys. GFR is typically expressed in units of ml/min:
  • GFR glomerular filtration rate
  • eGFR glomerular filtration rate
  • Creatinine clearance is used to measure GFR. Creatinine is produced naturally by the body (creatinine is a metabolite of creatine, which is found in muscle). It is freely filtered by the glomerulus, but also actively secreted by the renal tubules in very small amounts such that creatinine clearance overestimates actual GFR by 10-20%. This margin of error is acceptable considering the ease with which creatinine clearance is measured.
  • Creatinine clearance can be calculated if values for creatinine's urine concentration (UQ-), urine flow rate (V), and creatinine's plasma concentration (P Cr ) are known. Since the product of urine concentration and urine flow rate yields creatinine's excretion rate, creatinine clearance is also said to be its excretion rate (Uc r xV) divided by its plasma concentration. This is commonly represented mathematically as:
  • the CCr is often corrected for the body surface area (BSA) and expressed compared to the average sized man as ml/min/1.73 m2. While most adults have a BSA that approaches 1.7 (1.6-1.9), extremely obese or slim patients should have their CCr corrected for their actual BSA:
  • the clinician can readily select a treatment regimen that is compatible with the diagnosis, such as initiating renal replacement therapy, withdrawing delivery of compounds that are known to be damaging to the kidney, kidney transplantation, delaying or avoiding procedures that are known to be damaging to the kidney, modifying diuretic administration, initiating goal directed therapy, etc.
  • a treatment regimen that is compatible with the diagnosis, such as initiating renal replacement therapy, withdrawing delivery of compounds that are known to be damaging to the kidney, kidney transplantation, delaying or avoiding procedures that are known to be damaging to the kidney, modifying diuretic administration, initiating goal directed therapy, etc.
  • the skilled artisan is aware of appropriate treatments for numerous diseases discussed in relation to the methods of diagnosis described herein. See, e.g., Merck Manual of Diagnosis and Therapy, 17th Ed. Merck Research Laboratories, Whitehouse Station, NJ, 1999.
  • the markers of the present invention may be used to monitor a course of treatment. For example, improved or worsened prognostic state may indicate that a particular treatment is or is not eff
  • Example 1 Contrast-induced nephropathy sample collection
  • the objective of this sample collection study is to collect samples of plasma and urine and clinical data from patients before and after receiving intravascular contrast media.
  • Approximately 250 adults undergoing radiographic/angiographic procedures involving intravascular administration of iodinated contrast media are enrolled.
  • each patient must meet all of the following inclusion criteria and none of the following exclusion criteria: Inclusion Criteria males and females 18 years of age or older; undergoing a radiographic / angiographic procedure (such as a CT scan or coronary intervention) involving the intravascular administration of contrast media; expected to be hospitalized for at least 48 hours after contrast administration. able and willing to provide written informed consent for study participation and to comply with all study procedures.
  • Exclusion Criteria renal transplant recipients acutely worsening renal function prior to the contrast procedure; already receiving dialysis (either acute or chronic) or in imminent need of dialysis at enrollment; expected to undergo a major surgical procedure (such as involving cardiopulmonary bypass) or an additional imaging procedure with contrast media with significant risk for further renal insult within the 48 hrs following contrast administration; participation in an interventional clinical study with an experimental therapy within the previous 30 days; known infection with human immunodeficiency virus (HIV) or a hepatitis virus.
  • HAV human immunodeficiency virus
  • an EDTA anti-coagulated blood sample (10 mL) and a urine sample (10 mL) are collected from each patient. Blood and urine samples are then collected at 4 ( ⁇ 0.5), 8 ( ⁇ 1), 24 ( ⁇ 2) 48 ( ⁇ 2), and 72 ( ⁇ 2) hrs following the last administration of contrast media during the index contrast procedure. Blood is collected via direct venipuncture or via other available venous access, such as an existing femoral sheath, central venous line, peripheral intravenous line or hep-lock. These study blood samples are processed to plasma at the clinical site, frozen and shipped to Astute Medical, Inc., San Diego, CA. The study urine samples are frozen and shipped to Astute Medical, Inc.
  • Serum creatinine is assessed at the site immediately prior to the first contrast administration (after any pre-procedure hydration) and at 4 ( ⁇ 0.5), 8 ( ⁇ 1), 24 ( ⁇ 2) and 48 ( ⁇ 2) ), and 72 ( ⁇ 2) hours following the last administration of contrast (ideally at the same time as the study samples are obtained).
  • each patient's status is evaluated through day 30 with regard to additional serum and urine creatinine measurements, a need for dialysis, hospitalization status, and adverse clinical outcomes (including mortality).
  • Example 2 Cardiac surgery sample collection
  • the objective of this sample collection study is to collect samples of plasma and urine and clinical data from patients before and after undergoing cardiovascular surgery, a procedure known to be potentially damaging to kidney function. Approximately 900 adults undergoing such surgery are enrolled. To be enrolled in the study, each patient must meet all of the following inclusion criteria and none of the following exclusion criteria:
  • HAV human immunodeficiency virus
  • an EDTA anti-coagulated blood sample (10 mL), whole blood (3 mL), and a urine sample (35 mL) are collected from each patient. Blood and urine samples are then collected at 3 ( ⁇ 0.5), 6 ( ⁇ 0.5), 12 ( ⁇ 1), 24 ( ⁇ 2) and 48 ( ⁇ 2) hrs following the procedure and then daily on days 3 through 7 if the subject remains in the hospital. Blood is collected via direct venipuncture or via other available venous access, such as an existing femoral sheath, central venous line, peripheral intravenous line or hep-lock.
  • These study blood samples are frozen and shipped to Astute Medical, Inc., San Diego, CA.
  • the study urine samples are frozen and shipped to Astute Medical, Inc.
  • Example 3 Acutely ill subject sample collection
  • the objective of this study is to collect samples from acutely ill patients. Approximately 900 adults expected to be in the ICU for at least 48 hours will be enrolled. To be enrolled in the study, each patient must meet all of the following inclusion criteria and none of the following exclusion criteria:
  • Study population 1 approximately 300 patients that have at least one of: shock (SBP ⁇ 90 mmHg and/or need for vasopressor support to maintain MAP > 60 mmHg and/or documented drop in SBP of at least 40 mmHg); and sepsis;
  • shock SBP ⁇ 90 mmHg and/or need for vasopressor support to maintain MAP > 60 mmHg and/or documented drop in SBP of at least 40 mmHg
  • sepsis sepsis
  • Study population 2 approximately 300 patients that have at least one of: IV antibiotics ordered in computerized physician order entry (CPOE) within 24 hours of enrollment; contrast media exposure within 24 hours of enrollment; increased Intra- Abdominal Pressure with acute decompensated heart failure; and severe trauma as the primary reason for ICU admission and likely to be hospitalized in the ICU for 48 hours after enrollment;
  • IV antibiotics ordered in computerized physician order entry CPOE
  • contrast media exposure within 24 hours of enrollment
  • severe trauma as the primary reason for ICU admission and likely to be hospitalized in the ICU for 48 hours after enrollment
  • a known risk factor for acute renal injury e.g.
  • HAV human immunodeficiency virus
  • an EDTA anti-coagulated blood sample (10 mL) and a urine sample (25-30 mL) are collected from each patient. Blood and urine samples are then collected at 4 ( ⁇ 0.5) and 8 ( ⁇ 1) hours after contrast administration (if applicable); at 12 ( ⁇ 1), 24 ( ⁇ 2), and 48 ( ⁇ 2) hours after enrollment, and thereafter daily up to day 7 to day 14 while the subject is hospitalized. Blood is collected via direct venipuncture or via other available venous access, such as an existing femoral sheath, central venous line, peripheral intravenous line or hep-lock. These study blood samples are processed to plasma at the clinical site, frozen and shipped to Astute Medical, Inc., San Diego, CA. The study urine samples are frozen and shipped to Astute Medical, Inc.
  • Analytes are is measured using standard sandwich enzyme immunoassay techniques.
  • a first antibody which binds the analyte is immobilized in wells of a 96 well polystyrene microplate.
  • Analyte standards and test samples are pipetted into the appropriate wells and any analyte present is bound by the immobilized antibody.
  • a horseradish peroxidase-conjugated second antibody which binds the analyte is added to the wells, thereby forming sandwich complexes with the analyte (if present) and the first antibody.
  • a substrate solution comprising tetramethylbenzidine and hydrogen peroxide is added to the wells. Color develops in proportion to the amount of analyte present in the sample. The color development is stopped and the intensity of the color is measured at 540 nm or 570 nm. An analyte concentration is assigned to the test sample by comparison to a standard curve determined from the analyte standards.
  • Concentrations are expressed in the following examples as follows: soluble p- selectin - ng/mL; protein NOV homolog - pg.mL; netrin 4 - ng/mL; haptoglobin - mg/mL; alpha- 1 -antitrypsin - mg/mL; leukocyte elastase - ng/mL; soluble tumor necrosis factor receptor superfamily member 6 - pg/mL; soluble tumor necrosis factor ligand superfamily member 6 - pg/mL; soluble intercellular adhesion molecule 2 - units/mL; caspase 3 (active) - ng/mL; soluble platelet endothelial cell adhesion molecule - ng/mL; heat shock protein beta-1 - ng/mL; soluble epidermal growth factor receptor - pg/mL.
  • Two cohorts were defined as (Cohort 1) patients that did not progress beyond stage 0, and (Cohort 2) patients that reached stage R, I, or F within 10 days.
  • marker levels in urine samples collected for Cohort 1. Marker concentrations were measured in urine samples collected from a subject at 0, 24 hours, and 48 hours prior to reaching stage R, I or F in Cohort 2.
  • the time "prior max stage” represents the time at which a sample is collected, relative to the time a particular patient reaches the lowest disease stage as defined for that cohort, binned into three groups which are +/- 12 hours. For example, 24 hr prior for this example (0 vs R, I, F) would mean 24 hr (+/- 12 hours) prior to reaching stage R (or I if no sample at R, or F if no sample at R or I).
  • Each marker was measured by standard immunoassay methods using commercially available assay reagents.
  • a receiver operating characteristic (ROC) curve was generated for each marker and the area under each ROC curve (AUC) was determined.
  • Patients in Cohort 2 were also separated according to the reason for adjudication to stage R, I, or F as being based on serum creatinine measurements (sCr), being based on urine output (UO), or being based on either serum creatinine measurements or urine output.
  • the stage 0 cohort may have included patients adjudicated to stage R, I, or F on the basis of urine output; for those patients adjudicated to stage R, I, or F on the basis of urine output alone, the stage 0 cohort may have included patients adjudicated to stage R, I, or F on the basis of serum creatinine measurements; and for those patients adjudicated to stage R, I, or F on the basis of serum creatinine measurements or urine output, the stage 0 cohort contains only patients in stage 0 for both serum creatinine measurements and urine output. Also, for those patients adjudicated to stage R, I, or F on the basis of serum creatinine measurements or urine output, the adjudication method which yielded the most severe RIFLE stage was used.
  • Cohort 1 2 Cohort 1 2 Cohort 1 Cohort 2 median 0 .244 0.183 0.244 0.290 0.244 0 000 average 0 .243 0.217 0.243 3.764 0.243 0 190 stdev 0 .153 0.162 0.153 16.703 0.153 na p (t-test) 0.527 0.134 na min 0 .000 0.000 0.000 0.000 0.000 0 190 max 0 .662 0.662 0.662 82.167 0.662 0 190 n (Samp) 51 22 51 24 51 1 n (Pat) 40 22 40 24 40 1
  • Protein NOV homolog sCr or UO
  • Cohort 1 Cohort 2 Cohort 1 Cohort 2 Cohort 1 Cohort 2 Cohort 1 Cohort 2 median 0.000 0.000 0.000 0.000 0.000 average 0.001 0.000 0.001 0.000 0.001 0.000 stdev 0.001 0.000 0.001 0.001 0.000 p (t-test) 0.403 0.705 0.560 min 0.000 0.000 0.000 0.000 0.000 max 0.007 0.002 0.007 0.003 0.007 0.001 n (Samp) 375 16 375 21 375 11 n (Pat) 127 16 127 21 127 11
  • Cohort 1 ( Cohort 2 Cohort 1 Cohort 2 Cohort 1 Cohort 2 median 0 .001 0.001 0.001 0.001 0.001 average 0 .001 0.001 0.001 0.001 0.001 stdev 0 .001 0.001 0.001 0.001 0.001 0.001 p (t-test) 0.093 0.063 0.261 min 0 .000 0.000 0.000 0.000 0.000 0.000 max 0 .005 0.002 0.005 0.003 0.005 0.003 n (Samp) 375 16 375 21 375 11 n (Pat) 127 16 127 21 127 11
  • Leukocyte elastase sCr or
  • Soluble tumor necrosis factor receptor superfamilv member 6 sCr only
  • Soluble tumor necrosis factor ligand superfamilv member 6 sCr only
  • Soluble intercellular adhesion molecule 2 sCr only
  • Soluble platelet endothelial cell adhesion molecule sCr only
  • Heat shock protein beta- 1 sCr only
  • Soluble epidermal growth factor receptor sCr only
  • Leukocyte elastase Leukocyte elastase
  • Soluble tumor necrosis factor receptor superfamily member 6 is Soluble tumor necrosis factor receptor superfamily member 6:
  • Soluble tumor necrosis factor ligand superfamily member 6 Soluble tumor necrosis factor ligand superfamily member 6:
  • Soluble intercellular adhesion molecule 2 Soluble intercellular adhesion molecule 2:
  • Soluble platelet endothelial cell adhesion molecule Soluble platelet endothelial cell adhesion molecule:
  • Heat shock protein beta- 1 Heat shock protein beta- 1 :
  • Soluble epidermal growth factor receptor Soluble epidermal growth factor receptor
  • Soluble p-selectin sCr or UO
  • Protein NOV homolog sCr or UO
  • Netrin 4 sCr or UO sCr only
  • Haptoglobin sCr or UO
  • Alpha- 1 -antitrypsin sCr or UO sCr onl
  • Leukocyte elastase sCr or UO
  • Soluble tumor necrosis factor ligand superfamilv member 6 sCr or UO
  • Soluble intercellular adhesion molecule 2 sCr or UO
  • Soluble platelet endothelial cell adhesion molecule sCr or UO
  • Heat shock protein beta- 1 sCr or UO
  • Soluble epidermal growth factor receptor sCr or UO
  • Example 7 Kidney injury markers for evaluating renal status in patients at
  • Protein NOV homolog sCr only
  • Alpha- 1 -antitrypsin sCr or UO
  • Leukocyte elastase sCr or UO
  • Soluble tumor necrosis factor receptor superfamilv member 6 sCr only
  • Soluble tumor necrosis factor ligand superfamilv member 6 sCr only
  • Soluble intercellular adhesion molecule 2 sCr only
  • Heat shock protein beta- 1 sCr only
  • Leukocyte elastase Leukocyte elastase
  • Soluble tumor necrosis factor receptor superfamily member 6 is Soluble tumor necrosis factor receptor superfamily member 6:
  • Soluble tumor necrosis factor ligand superfamily member 6 Soluble tumor necrosis factor ligand superfamily member 6:
  • Soluble intercellular adhesion molecule 2 Soluble intercellular adhesion molecule 2:
  • Heat shock protein beta- 1 Heat shock protein beta- 1 :
  • Leukocyte elastase sCr or UO
  • Soluble tumor necrosis factor ligand superfamilv member 6 sCr or UO
  • Heat shock protein beta- 1 sCr or UO
  • Example 8 Kidney injury markers for evaluating renal status in patients progressing from Stage R to Stages I and F
  • Soluble tumor necrosis factor receptor superfamily member 6 is Soluble tumor necrosis factor receptor superfamily member 6:
  • Leukocyte elastase Leukocyte elastase
  • Soluble tumor necrosis factor receptor superfamily member 6 is Soluble tumor necrosis factor receptor superfamily member 6:
  • Alpha- 1 -antitrypsin sCr or UO
  • Leukocyte elastase sCr or UO sCr only
  • Soluble tumor necrosis factor receptor superfamilv member 6 sCr or UO
  • Two cohorts were defined as (Cohort 1) patients that did not progress beyond stage 0, and (Cohort 2) patients that reached stage R, I, or F within 10 days.
  • marker levels in blood samples collected for Cohort 1. Marker concentrations were measured in the plasma component of the blood samples collected from a subject at 0, 24 hours, and 48 hours prior to reaching stage R, I or F in Cohort 2.
  • the time "prior max stage” represents the time at which a sample is collected, relative to the time a particular patient reaches the lowest disease stage as defined for that cohort, binned into three groups which are +/- 12 hours. For example, 24 hr prior for this example (0 vs R, I, F) would mean 24 hr (+/- 12 hours) prior to reaching stage R (or I if no sample at R, or F if no sample at R or I).
  • Each marker was measured by standard immunoassay methods using commercially available assay reagents.
  • a receiver operating characteristic (ROC) curve was generated for each marker and the area under each ROC curve (AUC) was determined.
  • Patients in Cohort 2 were also separated according to the reason for adjudication to stage R, I, or F as being based on serum creatinine measurements (sCr), being based on urine output (UO), or being based on either serum creatinine measurements or urine output.
  • the stage 0 cohort may have included patients adjudicated to stage R, I, or F on the basis of urine output; for those patients adjudicated to stage R, I, or F on the basis of urine output alone, the stage 0 cohort may have included patients adjudicated to stage R, I, or F on the basis of serum creatinine measurements; and for those patients adjudicated to stage R, I, or F on the basis of serum creatinine measurements or urine output, the stage 0 cohort contains only patients in stage 0 for both serum creatinine measurements and urine output.
  • Cohort 1 Cohort 2 Cohort 1 Cohort 2 Cohort 1 Cohort 2 Cohort 1 Cohort 2 median 2.465 1.640 2.465 1.910 2.465 1.985 average 2.759 2.213 2.759 2.473 2.759 1.804 stdev 2.083 2.136 2.083 2.067 2.083 0.796 p (t-test) 0.306 0.540 0.149 min 0.000 0.000 0.000 0.000 0.000 0.703 max 12.700 8.320 12.700 8.870 12.700 2.730 n (Samp) 386 16 386 21 386 10 n (Pat) 129 16 129 21 129 10
  • Leukocyte elastase sCr only
  • Soluble tumor necrosis factor receptor superfamily member 6 is Soluble tumor necrosis factor receptor superfamily member 6:
  • Soluble tumor necrosis factor ligand superfamily member 6 sCr only
  • Soluble intercellular adhesion molecule 2 Soluble intercellular adhesion molecule 2:
  • Soluble platelet endothelial cell adhesion molecule Soluble platelet endothelial cell adhesion molecule:
  • Heat shock protein beta- 1 Heat shock protein beta- 1 :
  • Leukocyte elastase Leukocyte elastase
  • Soluble tumor necrosis factor receptor superfamily member 6 is Soluble tumor necrosis factor receptor superfamily member 6:
  • Soluble tumor necrosis factor ligand superfamily member 6 Soluble tumor necrosis factor ligand superfamily member 6:
  • Soluble intercellular adhesion molecule 2 Soluble intercellular adhesion molecule 2:
  • Soluble platelet endothelial cell adhesion molecule Soluble platelet endothelial cell adhesion molecule:
  • Heat shock protein beta- 1 Heat shock protein beta- 1 :
  • Protein NOV homolog sCr or UO
  • sCr only Netrin 4 sCr or UO sCr only
  • Haptoglobin sCr or UO
  • Alpha- 1 -antitrypsin sCr or UO
  • Leukocyte elastase sCr or UO
  • Soluble tumor necrosis factor ligand superfamilv member 6 sCr or UO
  • Soluble intercellular adhesion molecule 2 sCr or UO sCr only
  • Soluble platelet endothelial cell adhesion molecule sCr or UO
  • Heat shock protein beta- 1 sCr or UO sCr onl
  • Example 10 Kidney injury markers for evaluating renal status in patients at
  • Protein NOV homolog sCr or UO
  • Cohort 1 Cohort 2 Cohort 1 Cohort 2 Cohort 1 Cohort 2 Cohort 1 Cohort 2 median 0.014 0.037 0.014 0 100 0.014 0.020 average 0.200 0.037 0.200 0 100 0.200 0.020 stdev 1.212 0.022 1.212 0 086 1.212 0.004 p (t-test) 0.850 0 908 0.835
  • Alpha- 1 -antitrypsin sCr or UO
  • Leukocyte elastase Leukocyte elastase
  • Soluble tumor necrosis factor receptor superfamily member 6 sCr only Soluble tumor necrosis factor ligand superfamily member 6: sCr only
  • Soluble intercellular adhesion molecule 2 sCr only Heat shock protein beta- 1 :
  • Soluble epidermal growth factor receptor Soluble epidermal growth factor receptor

Abstract

The present invention relates to methods and compositions for monitoring, diagnosis, prognosis, and determination of treatment regimens in subjects suffering from or suspected of having a renal injury. In particular, the invention relates to using assays that detect one or more markers selected from the group consisting of soluble p-selectin, protein NOV homolog, soluble epidermal growth factor receptor, netrin-4, haptoglobin, heat shock protein beta-1, alpha- 1 -antitrypsin, leukocyte elastase, soluble tumor necrosis factor receptor superfamily member 6, soluble tumor necrosis factor ligand superfamily member 6, soluble intercellular adhesion molecule 2, active caspase-3, and soluble platelet endothelial cell adhesion molecule as diagnostic and prognostic biomarkers in renal injuries.

Description

METHODS AND COMPOSITIONS FOR DIAGNOSIS AND PROGNOSIS OF RENAL
INJURY AND RENAL FAILURE
[0001] The present invention claims priority from U.S. Provisional Patent Application 61/092,733 filed August 28, 2008, U.S. Provisional Patent Application 61/092,905 filed August 29, 2008, U.S. Provisional Patent Application 61/092,912 filed August 29, 2008, U.S. Provisional Patent Application 61/092,926 filed August 29, 2008, U.S. Provisional Patent Application 61/093,154 filed August 29, 2008, U.S. Provisional Patent Application 61/093,247 filed August 29, 2008, U.S. Provisional Patent Application 61/093,249 filed August 29, 2008, U.S. Provisional Patent Application 61/093,262 filed August 29, 2008, U.S. Provisional Patent Application 61/093,263 filed August 29, 2008, U.S. Provisional Patent Application 61/093,264 filed August 29, 2008, U.S. Provisional Patent Application 61/093,266 filed August 29, 2008, U.S. Provisional Patent Application 61/093,244 filed August 29, 2008, and U.S. Provisional Patent Application 61/093,272 filed August 29, 2008, each of which is hereby incorporated in its entirety including all tables, figures, and claims.
BACKGROUND OF THE INVENTION
[0002] The following discussion of the background of the invention is merely provided to aid the reader in understanding the invention and is not admitted to describe or constitute prior art to the present invention.
[0003] The kidney is responsible for water and solute excretion from the body. Its functions include maintenance of acid-base balance, regulation of electrolyte concentrations, control of blood volume, and regulation of blood pressure. As such, loss of kidney function through injury and/or disease results in substantial morbidity and mortality. A detailed discussion of renal injuries is provided in Harrison's Principles of Internal Medicine, 17th Ed., McGraw Hill, New York, pages 1741-1830, which are hereby incorporated by reference in their entirety. Renal disease and/or injury may be acute or chronic. Acute and chronic kidney disease are described as follows (from Current Medical Diagnosis & Treatment 2008, 47th Ed, McGraw Hill, New York, pages 785-815, which are hereby incorporated by reference in their entirety): "Acute renal failure is worsening of renal function over hours to days, resulting in the retention of nitrogenous wastes (such as urea nitrogen) and creatinine in the blood. Retention of these substances is called azotemia. Chronic renal failure (chronic kidney disease) results from an abnormal loss of renal function over months to years". [0004] Acute renal failure (ARF, also known as acute kidney injury, or AKI) is an abrupt (typically detected within about 48 hours to 1 week)reduction in glomerular filtration. This loss of filtration capacity results in retention of nitrogenous (urea and creatinine) and non- nitrogenous waste products that are normally excreted by the kidney, a reduction in urine output, or both. It is reported that ARF complicates about 5% of hospital admissions, 4-15% of cardiopulmonary bypass surgeries, and up to 30% of intensive care admissions. ARF may be categorized as prerenal, intrinsic renal, or postrenal in causation. Intrinsic renal disease can be further divided into glomerular, tubular, interstitial, and vascular abnormalities. Major causes of ARF are described in the following table, which is adapted from the Merck Manual, 17th ed., Chapter 222, and which is hereby incorporated by reference in their entirety:
Figure imgf000003_0001
Figure imgf000004_0001
[0005] In the case of ischemic ARF, the course of the disease may be divided into four phases. During an initiation phase, which lasts hours to days, reduced perfusion of the kidney is evolving into injury. Glomerular ultrafiltration reduces, the flow of filtrate is reduced due to debris within the tubules, and back leakage of filtrate through injured epithelium occurs. Renal injury can be mediated during this phase by reperfusion of the kidney. Initiation is followed by an extension phase which is characterized by continued ischemic injury and inflammation and may involve endothelial damage and vascular congestion. During the maintenance phase, lasting from 1 to 2 weeks, renal cell injury occurs, and glomerular filtration and urine output reaches a minimum. A recovery phase can follow in which the renal epithelium is repaired and GFR gradually recovers. Despite this, the survival rate of subjects with ARF may be as low as about 60%.
[0006] Acute kidney injury caused by radiocontrast agents (also called contrast media) and other nephrotoxins such as cyclosporine, antibiotics including aminoglycosides and anticancer drugs such as cisplatin manifests over a period of days to about a week. Contrast induced nephropathy (CIN, which is AKI caused by radiocontrast agents) is thought to be caused by intrarenal vasoconstriction (leading to ischemic injury) and from the generation of reactive oxygen species that are directly toxic to renal tubular epithelial cells. CIN classically presents as an acute (onset within 24-48h) but reversible (peak 3-5 days, resolution within 1 week) rise in blood urea nitrogen and serum creatinine.
[0007] A commonly reported criteria for defining and detecting AKI is an abrupt (typically within about 2-7 days or within a period of hospitalization) elevation of serum creatinine. Although the use of serum creatinine elevation to define and detect AKI is well established, the magnitude of the serum creatinine elevation and the time over which it is measured to define AKI varies considerably among publications. Traditionally, relatively large increases in serum creatinine such as 100%, 200%, an increase of at least 100% to a value over 2 mg/dL and other definitions were used to define AKI. However, the recent trend has been towards using smaller serum creatinine rises to define AKI. The relationship between serum creatinine rise, AKI and the associated health risks are reviewed in Praught and Shlipak, Curr Opin Nephrol Hypertens 14:265-270, 2005 and Chertow et al, J Am Soc Nephrol 16: 3365-3370, 2005, which, with the references listed therein, are hereby incorporated by reference in their entirety. As described in these publications, acute worsening renal function (AKI) and increased risk of death and other detrimental outcomes are now known to be associated with very small increases in serum creatinine. These increases may be determined as a relative (percent) value or a nominal value. Relative increases in serum creatinine as small as 20% from the pre-injury value have been reported to indicate acutely worsening renal function (AKI) and increased health risk, but the more commonly reported value to define AKI and increased health risk is a relative increase of at least 25%. Nominal increases as small as 0.3 mg/dL, 0.2 mg/dL or even 0.1 mg/dL have been reported to indicate worsening renal function and increased risk of death. Various time periods for the serum creatinine to rise to these threshold values have been used to define AKI, for example, ranging from 2 days, 3 days, 7 days, or a variable period defined as the time the patient is in the hospital or intensive care unit. These studies indicate there is not a particular threshold serum creatinine rise (or time period for the rise) for worsening renal function or AKI, but rather a continuous increase in risk with increasing magnitude of serum creatinine rise.
[0008] One study (Lassnigg et all, J Am Soc Nephrol 15:1597-1605, 2004, hereby incorporated by reference in its entirety) investigated both increases and decreases in serum creatinine. Patients with a mild fall in serum creatinine of -0.1 to -0.3 mg/dL following heart surgery had the lowest mortality rate. Patients with a larger fall in serum creatinine (more than or equal to -0.4 mg/dL) or any increase in serum creatinine had a larger mortality rate. These findings caused the authors to conclude that even very subtle changes in renal function (as detected by small creatinine changes within 48 hours of surgery) seriously effect patient's outcomes. In an effort to reach consensus on a unified classification system for using serum creatinine to define AKI in clinical trials and in clinical practice, Bellomo et al., Crit Care. 8(4):R204-12, 2004, which is hereby incorporated by reference in its entirety, proposes the following classifications for stratifying AKI patients:
"Risk": serum creatinine increased 1.5 fold from baseline OR urine production of <0.5 ml/kg body weight/hr for 6 hours;
"Injury": serum creatinine increased 2.0 fold from baseline OR urine production <0.5 ml/kg/hr for 12 h;
"Failure": serum creatinine increased 3.0 fold from baseline OR creatinine >355 μmol/1 (with a rise of >44) or urine output below 0.3 ml/kg/hr for 24 h or anuria for at least 12 hours;
And included two clinical outcomes:
"Loss": persistent need for renal replacement therapy for more than four weeks.
"ESRD": end stage renal disease — the need for dialysis for more than 3 months.
These criteria are called the RIFLE criteria, which provide a useful clinical tool to classify renal status. As discussed in Kellum, Crit. Care Med. 36: S 141-45, 2008 and Ricci et al, Kidney Int. 73, 538-546, 2008, each hereby incorporated by reference in its entirety, the RIFLE criteria provide a uniform definition of AKI which has been validated in numerous studies.
[0009] More recently, Mehta et al, Crit. Care 11:R31 (doi:10.1186.cc5713), 2007, hereby incorporated by reference in its entirety, proposes the following similar classifications for stratifying AKI patients, which have been modified from RIFLE:
"Stage I": increase in serum creatinine of more than or equal to 0.3 mg/dL (> 26.4 μmol/L) or increase to more than or equal to 150% (1.5-fold) from baseline OR urine output less than 0.5 mL/kg per hour for more than 6 hours;
"Stage II": increase in serum creatinine to more than 200% (> 2-fold) from baseline OR urine output less than 0.5 mL/kg per hour for more than 12 hours; "Stage III": increase in serum creatinine to more than 300% (> 3-fold) from baseline OR serum creatinine > 354 μmol/L accompanied by an acute increase of at least 44 μmol/L OR urine output less than 0.3 mL/kg per hour for 24 hours or anuria for 12 hours.
[0010] The CIN Consensus Working Panel (McCollough et al, Rev Cardiovasc Med. 2006;7(4): 177-197, hereby incorporated by reference in its entirety) uses a serum creatinine rise of 25% to define Contrast induced nephropathy (which is a type of AKI).Although various groups propose slightly different criteria for using serum creatinine to detect AKI, the consensus is that small changes in serum creatinine, such as 0.3 mg/dL or 25%, are sufficient to detect AKI (worsening renal function) and that the magnitude of the serum creatinine change is an indicator of the severity of the AKI and mortality risk.
[0011] Although serial measurement of serum creatinine over a period of days is an accepted method of detecting and diagnosing AKI and is considered one of the most important tools to evaluate AKI patients, serum creatinine is generally regarded to have several limitations in the diagnosis, assessment and monitoring of AKI patients. The time period for serum creatinine to rise to values (e.g., a 0.3 mg/dL or 25% rise) considered diagnostic for AKI can be 48 hours or longer depending on the definition used. Since cellular injury in AKI can occur over a period of hours, serum creatinine elevations detected at 48 hours or longer can be a late indicator of injury, and relying on serum creatinine can thus delay diagnosis of AKI. Furthermore, serum creatinine is not a good indicator of the exact kidney status and treatment needs during the most acute phases of AKI when kidney function is changing rapidly. Some patients with AKI will recover fully, some will need dialysis (either short term or long term) and some will have other detrimental outcomes including death, major adverse cardiac events and chronic kidney disease. Because serum creatinine is a marker of filtration rate, it does not differentiate between the causes of AKI (pre-renal, intrinsic renal, post-renal obstruction, atheroembolic, etc) or the category or location of injury in intrinsic renal disease (for example, tubular, glomerular or interstitial in origin). Urine output is similarly limited, Knowing these things can be of vital importance in managing and treating patients with AKI.
[0012] These limitations underscore the need for better methods to detect and assess AKI, particularly in the early and subclinical stages, but also in later stages when recovery and repair of the kidney can occur. Furthermore, there is a need to better identify patients who are at risk of having an AKI. BRIEF SUMMARY OF THE INVENTION
[0013] It is an object of the invention to provide methods and compositions for evaluating renal function in a subject. As described herein, measurement of one or more markers selected from the group consisting of soluble p-selectin, protein NOV homolog, soluble epidermal growth factor receptor, netrin-4, haptoglobin, heat shock protein beta-1, alpha- 1 -antitrypsin, leukocyte elastase, soluble tumor necrosis factor receptor superfamily member 6, soluble tumor necrosis factor ligand superfamily member 6, soluble intercellular adhesion molecule 2, caspase-3 (and most preferably active caspase-3), and soluble platelet endothelial cell adhesion molecule (collectively referred to herein as "kidney injury markers, and individually as a "kidney injury marker") can be used for diagnosis, prognosis, risk stratification, staging, monitoring, categorizing and determination of further diagnosis and treatment regimens in subjects suffering or at risk of suffering from an injury to renal function, reduced renal function, and/or acute renal failure (also called acute kidney injury).
[0014] These kidney injury markers may be used, individually or in panels comprising a plurality of kidney injury markers, for risk stratification (that is, to identify subjects at risk for a future injury to renal function, for future progression to reduced renal function, for future progression to ARF, for future improvement in renal function, etc.); for diagnosis of existing disease (that is, to identify subjects who have suffered an injury to renal function, who have progressed to reduced renal function, who have progressed to ARF, etc.); for monitoring for deterioration or improvement of renal function; and for predicting a future medical outcome, such as improved or worsening renal function, a decreased or increased mortality risk, a decreased or increased risk that a subject will require renal replacement therapy (i.e., hemodialysis, peritoneal dialysis, hemofiltration, and/or renal transplantation, a decreased or increased risk that a subject will recover from an injury to renal function, a decreased or increased risk that a subject will recover from ARF, a decreased or increased risk that a subject will progress to end stage renal disease, a decreased or increased risk that a subject will progress to chronic renal failure, a decreased or increased risk that a subject will suffer rejection of a transplanted kidney, etc.
[0015] In a first aspect, the present invention relates to methods for evaluating renal status in a subject. These methods comprise performing an assay method that is configured to detect one or more kidney injury markers of the present invention in a body fluid sample obtained from the subject. The assay result(s), for example a measured concentration of one or more markers selected from the group consisting of soluble p-selectin, protein NOV homolog, soluble epidermal growth factor receptor, netrin-4, haptoglobin, heat shock protein beta-1, alpha- 1 -antitrypsin, leukocyte elastase, soluble tumor necrosis factor receptor superfamily member 6, soluble tumor necrosis factor ligand superfamily member 6, soluble intercellular adhesion molecule 2, caspase-3 (and most preferably active caspase-3), and soluble platelet endothelial cell adhesion molecule is/are then correlated to the renal status of the subject. This correlation to renal status may include correlating the assay result(s) to one or more of risk stratification, diagnosis, prognosis, staging, classifying and monitoring of the subject as described herein. Thus, the present invention utilizes one or more kidney injury markers of the present invention for the evaluation of renal injury.
[0016] In certain embodiments, the methods for evaluating renal status described herein are methods for risk stratification of the subject; that is, assigning a likelihood of one or more future changes in renal status to the subject. In these embodiments, the assay result(s) is/are correlated to one or more such future changes. The following are preferred risk stratification embodiments.
[0017] In preferred risk stratification embodiments, these methods comprise determining a subject's risk for a future injury to renal function, and the assay result(s) is/are correlated to a likelihood of such a future injury to renal function. For example, the measured concentration(s) may each be compared to a threshold value. For a "positive going" kidney injury marker, an increased likelihood of suffering a future injury to renal function is assigned to the subject when the measured concentration is above the threshold, relative to a likelihood assigned when the measured concentration is below the threshold. For a "negative going" kidney injury marker, an increased likelihood of suffering a future injury to renal function is assigned to the subject when the measured concentration is below the threshold, relative to a likelihood assigned when the measured concentration is above the threshold.
[0018] In other preferred risk stratification embodiments, these methods comprise determining a subject's risk for future reduced renal function, and the assay result(s) is/are correlated to a likelihood of such reduced renal function. For example, the measured concentrations may each be compared to a threshold value. For a "positive going" kidney injury marker, an increased likelihood of suffering a future reduced renal function is assigned to the subject when the measured concentration is above the threshold, relative to a likelihood assigned when the measured concentration is below the threshold. For a "negative going" kidney injury marker, an increased likelihood of future reduced renal function is assigned to the subject when the measured concentration is below the threshold, relative to a likelihood assigned when the measured concentration is above the threshold.
[0019] In still other preferred risk stratification embodiments, these methods comprise determining a subject's likelihood for a future improvement in renal function, and the assay result(s) is/are correlated to a likelihood of such a future improvement in renal function. For example, the measured concentration(s) may each be compared to a threshold value. For a "positive going" kidney injury marker, an increased likelihood of a future improvement in renal function is assigned to the subject when the measured concentration is below the threshold, relative to a likelihood assigned when the measured concentration is above the threshold. For a "negative going" kidney injury marker, an increased likelihood of a future improvement in renal function is assigned to the subject when the measured concentration is above the threshold, relative to a likelihood assigned when the measured concentration is below the threshold.
[0020] In yet other preferred risk stratification embodiments, these methods comprise determining a subject's risk for progression to ARF, and the result(s) is/are correlated to a likelihood of such progression to ARF. For example, the measured concentration(s) may each be compared to a threshold value. For a "positive going" kidney injury marker, an increased likelihood of progression to ARF is assigned to the subject when the measured concentration is above the threshold, relative to a likelihood assigned when the measured concentration is below the threshold. For a "negative going" kidney injury marker, an increased likelihood of progression to ARF is assigned to the subject when the measured concentration is below the threshold, relative to a likelihood assigned when the measured concentration is above the threshold.
[0021] And in other preferred risk stratification embodiments, these methods comprise determining a subject's outcome risk, and the assay result(s) is/are correlated to a likelihood of the occurrence of a clinical outcome related to a renal injury suffered by the subject. For example, the measured concentration(s) may each be compared to a threshold value. For a "positive going" kidney injury marker, an increased likelihood of one or more of: acute kidney injury, progression to a worsening stage of AKI, mortality, a requirement for renal replacement therapy, a requirement for withdrawal of renal toxins, end stage renal disease, heart failure, stroke, myocardial infarction, progression to chronic kidney disease, etc., is assigned to the subject when the measured concentration is above the threshold, relative to a likelihood assigned when the measured concentration is below the threshold. For a "negative going" kidney injury marker, an increased likelihood of one or more of: acute kidney injury, progression to a worsening stage of AKI, mortality, a requirement for renal replacement therapy, a requirement for withdrawal of renal toxins, end stage renal disease, heart failure, stroke, myocardial infarction, progression to chronic kidney disease, etc., is assigned to the subject when the measured concentration is below the threshold, relative to a likelihood assigned when the measured concentration is above the threshold.
[0022] In such risk stratification embodiments, preferably the likelihood or risk assigned is that an event of interest is more or less likely to occur within 180 days of the time at which the body fluid sample is obtained from the subject. In particularly preferred embodiments, the likelihood or risk assigned relates to an event of interest occurring within a shorter time period such as 18 months, 120 days, 90 days, 60 days, 45 days, 30 days, 21 days, 14 days, 7 days, 5 days, 96 hours, 72 hours, 48 hours, 36 hours, 24 hours, 12 hours, or less. A risk at 0 hours of the time at which the body fluid sample is obtained from the subject is equivalent to diagnosis of a current condition.
[0023] In preferred risk stratification embodiments, the subject is selected for risk stratification based on the pre-existence in the subject of one or more known risk factors for prerenal, intrinsic renal, or postrenal ARF. For example, a subject undergoing or having undergone major vascular surgery, coronary artery bypass, or other cardiac surgery; a subject having pre-existing congestive heart failure, preeclampsia, eclampsia, diabetes mellitus, hypertension, coronary artery disease, proteinuria, renal insufficiency, glomerular filtration below the normal range, cirrhosis, serum creatinine above the normal range, or sepsis; or a subject exposed to NSAIDs, cyclosporines, tacrolimus, aminoglycosides, foscarnet, ethylene glycol, hemoglobin, myoglobin, ifosfamide, heavy metals, methotrexate, radiopaque contrast agents, or streptozotocin are all preferred subjects for monitoring risks according to the methods described herein. This list is not meant to be limiting. By "pre- existence" in this context is meant that the risk factor exists at the time the body fluid sample is obtained from the subject. In particularly preferred embodiments, a subject is chosen for risk stratification based on an existing diagnosis of injury to renal function, reduced renal function, or ARF.
[0024] In other embodiments, the methods for evaluating renal status described herein are methods for diagnosing a renal injury in the subject; that is, assessing whether or not a subject has suffered from an injury to renal function, reduced renal function, or ARF. In these embodiments, the assay result(s), for example a measured concentration of one or more markers selected from the group consisting of soluble p-selectin, protein NOV homolog, soluble epidermal growth factor receptor, netrin-4, haptoglobin, heat shock protein beta-1, alpha- 1 -antitrypsin, leukocyte elastase, soluble tumor necrosis factor receptor superfamily member 6, soluble tumor necrosis factor ligand superfamily member 6, soluble intercellular adhesion molecule 2, caspase-3 (and most preferably active caspase-3), and soluble platelet endothelial cell adhesion molecule is/are correlated to the occurrence or nonoccurrence of a change in renal status. The following are preferred diagnostic embodiments.
[0025] In preferred diagnostic embodiments, these methods comprise diagnosing the occurrence or nonoccurrence of an injury to renal function, and the assay result(s) is/are correlated to the occurrence or nonoccurrence of such an injury. For example, each of the measured concentration(s) may be compared to a threshold value. For a positive going marker, an increased likelihood of the occurrence of an injury to renal function is assigned to the subject when the measured concentration is above the threshold (relative to the likelihood assigned when the measured concentration is below the threshold); alternatively, when the measured concentration is below the threshold, an increased likelihood of the nonoccurrence of an injury to renal function may be assigned to the subject (relative to the likelihood assigned when the measured concentration is above the threshold). For a negative going marker, an increased likelihood of the occurrence of an injury to renal function is assigned to the subject when the measured concentration is below the threshold (relative to the likelihood assigned when the measured concentration is above the threshold); alternatively, when the measured concentration is above the threshold, an increased likelihood of the nonoccurrence of an injury to renal function may be assigned to the subject (relative to the likelihood assigned when the measured concentration is below the threshold).
[0026] In other preferred diagnostic embodiments, these methods comprise diagnosing the occurrence or nonoccurrence of reduced renal function, and the assay result(s) is/are correlated to the occurrence or nonoccurrence of an injury causing reduced renal function. For example, each of the measured concentration(s) may be compared to a threshold value. For a positive going marker, an increased likelihood of the occurrence of an injury causing reduced renal function is assigned to the subject when the measured concentration is above the threshold (relative to the likelihood assigned when the measured concentration is below the threshold); alternatively, when the measured concentration is below the threshold, an increased likelihood of the nonoccurrence of an injury causing reduced renal function may be assigned to the subject (relative to the likelihood assigned when the measured concentration is above the threshold). For a negative going marker, an increased likelihood of the occurrence of an injury causing reduced renal function is assigned to the subject when the measured concentration is below the threshold (relative to the likelihood assigned when the measured concentration is above the threshold); alternatively, when the measured concentration is above the threshold, an increased likelihood of the nonoccurrence of an injury causing reduced renal function may be assigned to the subject (relative to the likelihood assigned when the measured concentration is below the threshold).
[0027] In yet other preferred diagnostic embodiments, these methods comprise diagnosing the occurrence or nonoccurrence of ARF, and the assay result(s) is/are correlated to the occurrence or nonoccurrence of an injury causing ARF. For example, each of the measured concentration(s) may be compared to a threshold value. For a positive going marker, an increased likelihood of the occurrence of ARF is assigned to the subject when the measured concentration is above the threshold (relative to the likelihood assigned when the measured concentration is below the threshold); alternatively, when the measured concentration is below the threshold, an increased likelihood of the nonoccurrence of ARF may be assigned to the subject (relative to the likelihood assigned when the measured concentration is above the threshold). For a negative going marker, an increased likelihood of the occurrence of ARF is assigned to the subject when the measured concentration is below the threshold (relative to the likelihood assigned when the measured concentration is above the threshold); alternatively, when the measured concentration is above the threshold, an increased likelihood of the nonoccurrence of ARF may be assigned to the subject (relative to the likelihood assigned when the measured concentration is below the threshold).
[0028] In still other preferred diagnostic embodiments, these methods comprise diagnosing a subject as being in need of renal replacement therapy, and the assay result(s) is/are correlated to a need for renal replacement therapy. For example, each of the measured concentration(s) may be compared to a threshold value. For a positive going marker, an increased likelihood of the occurrence of an injury creating a need for renal replacement therapy is assigned to the subject when the measured concentration is above the threshold (relative to the likelihood assigned when the measured concentration is below the threshold); alternatively, when the measured concentration is below the threshold, an increased likelihood of the nonoccurrence of an injury creating a need for renal replacement therapy may be assigned to the subject (relative to the likelihood assigned when the measured concentration is above the threshold). For a negative going marker, an increased likelihood of the occurrence of an injury creating a need for renal replacement therapy is assigned to the subject when the measured concentration is below the threshold (relative to the likelihood assigned when the measured concentration is above the threshold); alternatively, when the measured concentration is above the threshold, an increased likelihood of the nonoccurrence of an injury creating a need for renal replacement therapy may be assigned to the subject (relative to the likelihood assigned when the measured concentration is below the threshold).
[0029] In still other preferred diagnostic embodiments, these methods comprise diagnosing a subject as being in need of renal transplantation, and the assay result(sθ is/are correlated to a need for renal transplantation. For example, each of the measured concentration(s) may be compared to a threshold value. For a positive going marker, an increased likelihood of the occurrence of an injury creating a need for renal transplantation is assigned to the subject when the measured concentration is above the threshold (relative to the likelihood assigned when the measured concentration is below the threshold); alternatively, when the measured concentration is below the threshold, an increased likelihood of the nonoccurrence of an injury creating a need for renal transplantation may be assigned to the subject (relative to the likelihood assigned when the measured concentration is above the threshold). For a negative going marker, an increased likelihood of the occurrence of an injury creating a need for renal transplantation is assigned to the subject when the measured concentration is below the threshold (relative to the likelihood assigned when the measured concentration is above the threshold); alternatively, when the measured concentration is above the threshold, an increased likelihood of the nonoccurrence of an injury creating a need for renal transplantation may be assigned to the subject (relative to the likelihood assigned when the measured concentration is below the threshold).
[0030] In still other embodiments, the methods for evaluating renal status described herein are methods for monitoring a renal injury in the subject; that is, assessing whether or not renal function is improving or worsening in a subject who has suffered from an injury to renal function, reduced renal function, or ARF. In these embodiments, the assay result(s), for example a measured concentration of one or more markers selected from the group consisting of soluble p-selectin, protein NOV homolog, soluble epidermal growth factor receptor, netrin-4, haptoglobin, heat shock protein beta-1, alpha- 1 -antitrypsin, leukocyte elastase, soluble tumor necrosis factor receptor superfamily member 6, soluble tumor necrosis factor ligand superfamily member 6, soluble intercellular adhesion molecule 2, caspase-3 (and most preferably active caspase-3), and soluble platelet endothelial cell adhesion molecule is/are correlated to the occurrence or nonoccurrence of a change in renal status. The following are preferred monitoring embodiments.
[0031] In preferred monitoring embodiments, these methods comprise monitoring renal status in a subject suffering from an injury to renal function, and the assay result(s) is/are correlated to the occurrence or nonoccurrence of a change in renal status in the subject. For example, the measured concentration(s) may be compared to a threshold value. For a positive going marker, when the measured concentration is above the threshold, a worsening of renal function may be assigned to the subject; alternatively, when the measured concentration is below the threshold, an improvement of renal function may be assigned to the subject. For a negative going marker, when the measured concentration is below the threshold, a worsening of renal function may be assigned to the subject; alternatively, when the measured concentration is above the threshold, an improvement of renal function may be assigned to the subject.
[0032] In other preferred monitoring embodiments, these methods comprise monitoring renal status in a subject suffering from reduced renal function, and the assay result(s) is/are correlated to the occurrence or nonoccurrence of a change in renal status in the subject. For example, the measured concentration(s) may be compared to a threshold value. For a positive going marker, when the measured concentration is above the threshold, a worsening of renal function may be assigned to the subject; alternatively, when the measured concentration is below the threshold, an improvement of renal function may be assigned to the subject. For a negative going marker, when the measured concentration is below the threshold, a worsening of renal function may be assigned to the subject; alternatively, when the measured concentration is above the threshold, an improvement of renal function may be assigned to the subject.
[0033] In yet other preferred monitoring embodiments, these methods comprise monitoring renal status in a subject suffering from acute renal failure, and the assay result(s) is/are correlated to the occurrence or nonoccurrence of a change in renal status in the subject. For example, the measured concentration(s) may be compared to a threshold value. For a positive going marker, when the measured concentration is above the threshold, a worsening of renal function may be assigned to the subject; alternatively, when the measured concentration is below the threshold, an improvement of renal function may be assigned to the subject. For a negative going marker, when the measured concentration is below the threshold, a worsening of renal function may be assigned to the subject; alternatively, when the measured concentration is above the threshold, an improvement of renal function may be assigned to the subject.
[0034] In other additional preferred monitoring embodiments, these methods comprise monitoring renal status in a subject at risk of an injury to renal function due to the pre- existence of one or more known risk factors for prerenal, intrinsic renal, or postrenal ARF, and the assay result(s) is/are correlated to the occurrence or nonoccurrence of a change in renal status in the subject. For example, the measured concentration(s) may be compared to a threshold value. For a positive going marker, when the measured concentration is above the threshold, a worsening of renal function may be assigned to the subject; alternatively, when the measured concentration is below the threshold, an improvement of renal function may be assigned to the subject. For a negative going marker, when the measured concentration is below the threshold, a worsening of renal function may be assigned to the subject; alternatively, when the measured concentration is above the threshold, an improvement of renal function may be assigned to the subject.
[0035] In still other embodiments, the methods for evaluating renal status described herein are methods for classifying a renal injury in the subject; that is, determining whether a renal injury in a subject is prerenal, intrinsic renal, or postrenal; and/or further subdividing these classes into subclasses such as acute tubular injury, acute glomerulonephritis acute tubulointerstitial nephritis, acute vascular nephropathy, or infiltrative disease; and/or assigning a likelihood that a subject will progress to a particular RIFLE stage. In these embodiments, the assay result(s), for example a measured concentration of one or more markers selected from the group consisting of soluble p-selectin, protein NOV homolog, soluble epidermal growth factor receptor, netrin-4, haptoglobin, heat shock protein beta-1, alpha- 1 -antitrypsin, leukocyte elastase, soluble tumor necrosis factor receptor superfamily member 6, soluble tumor necrosis factor ligand superfamily member 6, soluble intercellular adhesion molecule 2, caspase-3 (and most preferably active caspase-3), and soluble platelet endothelial cell adhesion molecule is/are correlated to a particular class and/or subclass. The following are preferred classification embodiments.
[0036] In preferred classification embodiments, these methods comprise determining whether a renal injury in a subject is prerenal, intrinsic renal, or postrenal; and/or further subdividing these classes into subclasses such as acute tubular injury, acute glomerulonephritis acute tubulointerstitial nephritis, acute vascular nephropathy, or infiltrative disease; and/or assigning a likelihood that a subject will progress to a particular RIFLE stage, and the assay result(s) is/are correlated to the injury classification for the subject. For example, the measured concentration may be compared to a threshold value, and when the measured concentration is above the threshold, a particular classification is assigned; alternatively, when the measured concentration is below the threshold, a different classification may be assigned to the subject.
[0037] A variety of methods may be used by the skilled artisan to arrive at a desired threshold value for use in these methods. For example, the threshold value may be determined from a population of normal subjects by selecting a concentration representing the 75th, 85th, 90th, 95th, or 99th percentile of a kidney injury marker measured in such normal subjects. Alternatively, the threshold value may be determined from a "diseased" population of subjects, e.g., those suffering from an injury or having a predisposition for an injury (e.g., progression to ARF or some other clinical outcome such as death, dialysis, renal transplantation, etc.), by selecting a concentration representing the 75th, 85th, 90th, 95th, or 99th percentile of a kidney injury marker measured in such subjects. In another alternative, the threshold value may be determined from a prior measurement of a kidney injury marker in the same subject; that is, a temporal change in the level of a kidney injury marker in the subject may be used to assign risk to the subject.
[001] The foregoing discussion is not meant to imply, however, that the kidney injury markers of the present invention must be compared to corresponding individual thresholds. Methods for combining assay results can comprise the use of multivariate logistical regression, loglinear modeling, neural network analysis, n-of-m analysis, decision tree analysis, calculating ratios of markers, etc. This list is not meant to be limiting. In these methods, a composite result which is determined by combining individual markers may be treated as if it is itself a marker; that is, a threshold may be determined for the composite result as described herein for individual markers, and the composite result for an individual patient compared to this threshold.
[0038] The ability of a particular test to distinguish two populations can be established using ROC analysis. For example, ROC curves established from a "first" subpopulation which is predisposed to one or more future changes in renal status, and a "second" subpopulation which is not so predisposed can be used to calculate a ROC curve, and the area under the curve provides a measure of the quality of the test. Preferably, the tests described herein provide a ROC curve area greater than 0.5, preferably at least 0.6, more preferably 0.7, still more preferably at least 0.8, even more preferably at least 0.9, and most preferably at least 0.95.
[0039] In certain aspects, the measured concentration of one or more kidney injury markers, or a composite of such markers, may be treated as continuous variables. For example, any particular concentration can be converted into a corresponding probability of a future reduction in renal function for the subject, the occurrence of an injury, a classification, etc. In yet another alternative, a threshold that can provide an acceptable level of specificity and sensitivity in separating a population of subjects into "bins" such as a "first" subpopulation (e.g., which is predisposed to one or more future changes in renal status, the occurrence of an injury, a classification, etc.) and a "second" subpopulation which is not so predisposed. A threshold value is selected to separate this first and second population by one or more of the following measures of test accuracy: an odds ratio greater than 1, preferably at least about 2 or more or about 0.5 or less, more preferably at least about 3 or more or about 0.33 or less, still more preferably at least about 4 or more or about 0.25 or less, even more preferably at least about 5 or more or about 0.2 or less, and most preferably at least about 10 or more or about 0.1 or less; a specificity of greater than 0.5, preferably at least about 0.6, more preferably at least about 0.7, still more preferably at least about 0.8, even more preferably at least about 0.9 and most preferably at least about 0.95, with a corresponding sensitivity greater than 0.2, preferably greater than about 0.3, more preferably greater than about 0.4, still more preferably at least about 0.5, even more preferably about 0.6, yet more preferably greater than about 0.7, still more preferably greater than about 0.8, more preferably greater than about 0.9, and most preferably greater than about 0.95; a sensitivity of greater than 0.5, preferably at least about 0.6, more preferably at least about 0.7, still more preferably at least about 0.8, even more preferably at least about 0.9 and most preferably at least about 0.95, with a corresponding specificity greater than 0.2, preferably greater than about 0.3, more preferably greater than about 0.4, still more preferably at least about 0.5, even more preferably about 0.6, yet more preferably greater than about 0.7, still more preferably greater than about 0.8, more preferably greater than about 0.9, and most preferably greater than about 0.95; at least about 75% sensitivity, combined with at least about 75% specificity; a positive likelihood ratio (calculated as sensitivity/(l-specificity)) of greater than 1, at least about 2, more preferably at least about 3, still more preferably at least about 5, and most preferably at least about 10; or a negative likelihood ratio (calculated as (l-sensitivity)/specificity) of less than 1, less than or equal to about 0.5, more preferably less than or equal to about 0.3, and most preferably less than or equal to about 0.1.
The term "about" in the context of any of the above measurements refers to +/- 5% of a given measurement.
[0040] Multiple thresholds may also be used to assess renal status in a subject. For example, a "first" subpopulation which is predisposed to one or more future changes in renal status, the occurrence of an injury, a classification, etc., and a "second" subpopulation which is not so predisposed can be combined into a single group. This group is then subdivided into three or more equal parts (known as tertiles, quartiles, quintiles, etc., depending on the number of subdivisions). An odds ratio is assigned to subjects based on which subdivision they fall into. If one considers a tertile, the lowest or highest tertile can be used as a reference for comparison of the other subdivisions. This reference subdivision is assigned an odds ratio of 1. The second tertile is assigned an odds ratio that is relative to that first tertile. That is, someone in the second tertile might be 3 times more likely to suffer one or more future changes in renal status in comparison to someone in the first tertile. The third tertile is also assigned an odds ratio that is relative to that first tertile.
[0041] In certain embodiments, the assay method is an immunoassay. Antibodies for use in such assays will specifically bind a full length kidney injury marker of interest, and may also bind one or more polypeptides that are "related" thereto, as that term is defined hereinafter. Numerous immunoassay formats are known to those of skill in the art. Preferred body fluid samples are selected from the group consisting of urine, blood, serum, saliva, tears, and plasma.
[0042] The foregoing method steps should not be interpreted to mean that the kidney injury marker assay result(s) is/are used in isolation in the methods described herein. Rather, additional variables or other clinical indicia may be included in the methods described herein. For example, a risk stratification, diagnostic, classification, monitoring, etc. method may combine the assay result(s) with one or more variables measured for the subject selected from the group consisting of demographic information (e.g., weight, sex, age, race), medical history (e.g., family history, type of surgery, pre-existing disease such as aneurism, congestive heart failure, preeclampsia, eclampsia, diabetes mellitus, hypertension, coronary artery disease, proteinuria, renal insufficiency, or sepsis, type of toxin exposure such as NSAIDs, cyclosporines, tacrolimus, aminoglycosides, foscarnet, ethylene glycol, hemoglobin, myoglobin, ifosfamide, heavy metals, methotrexate, radiopaque contrast agents, or strep tozotocin), clinical variables (e.g., blood pressure, temperature, respiration rate), risk scores (APACHE score, PREDICT score, TIMI Risk Score for UA/NSTEMI, Framingham Risk Score), a glomerular filtration rate, an estimated glomerular filtration rate, a urine production rate, a serum or plasma creatinine concentration, a urine creatinine concentration, a fractional excretion of sodium, a urine sodium concentration, a urine creatinine to serum or plasma creatinine ratio, a urine specific gravity, a urine osmolality, a urine urea nitrogen to plasma urea nitrogen ratio, a plasma BUN to creatnine ratio, a renal failure index calculated as urine sodium / (urine creatinine / plasma creatinine), a serum or plasma neutrophil gelatinase (NGAL) concentration, a urine NGAL concentration, a serum or plasma cystatin C concentration, a serum or plasma cardiac troponin concentration, a serum or plasma BNP concentration, a serum or plasma NTproBNP concentration, and a serum or plasma proBNP concentration. Other measures of renal function which may be combined with one or more kidney injury marker assay result(s) are described hereinafter and in Harrison's Principles of Internal Medicine, 17th Ed., McGraw Hill, New York, pages 1741-1830, and Current Medical Diagnosis & Treatment 2008, 47th Ed, McGraw Hill, New York, pages 785-815, each of which are hereby incorporated by reference in their entirety.
[0043] When more than one marker is measured, the individual markers may be measured in samples obtained at the same time, or may be determined from samples obtained at different (e.g., an earlier or later) times. The individual markers may also be measured on the same or different body fluid samples. For example, one kidney injury marker may be measured in a serum or plasma sample and another kidney injury marker may be measured in a urine sample. In addition, assignment of a likelihood may combine an individual kidney injury marker assay result with temporal changes in one or more additional variables.
[0044] In various related aspects, the present invention also relates to devices and kits for performing the methods described herein. Suitable kits comprise reagents sufficient for performing an assay for at least one of the described kidney injury markers, together with instructions for performing the described threshold comparisons. [0045] In certain embodiments, reagents for performing such assays are provided in an assay device, and such assay devices may be included in such a kit. Preferred reagents can comprise one or more solid phase antibodies, the solid phase antibody comprising antibody that detects the intended biomarker target(s) bound to a solid support. In the case of sandwich immunoassays, such reagents can also include one or more detectably labeled antibodies, the detectably labeled antibody comprising antibody that detects the intended biomarker target(s) bound to a detectable label. Additional optional elements that may be provided as part of an assay device are described hereinafter.
[0046] Detectable labels may include molecules that are themselves detectable (e.g., fluorescent moieties, electrochemical labels, eel (electrochemical luminescence) labels, metal chelates, colloidal metal particles, etc.) as well as molecules that may be indirectly detected by production of a detectable reaction product (e.g., enzymes such as horseradish peroxidase, alkaline phosphatase, etc.) or through the use of a specific binding molecule which itself may be detectable (e.g., a labeled antibody that binds to the second antibody, biotin, digoxigenin, maltose, oligohistidine, 2,4-dintrobenzene, phenylarsenate, ssDNA, dsDNA, etc.).
[0047] Generation of a signal from the signal development element can be performed using various optical, acoustical, and electrochemical methods well known in the art. Examples of detection modes include fluorescence, radiochemical detection, reflectance, absorbance, amperometry, conductance, impedance, interferometry, ellipsometry, etc. In certain of these methods, the solid phase antibody is coupled to a transducer (e.g., a diffraction grating, electrochemical sensor, etc) for generation of a signal, while in others, a signal is generated by a transducer that is spatially separate from the solid phase antibody (e.g., a fluorometer that employs an excitation light source and an optical detector). This list is not meant to be limiting. Antibody-based biosensors may also be employed to determine the presence or amount of analytes that optionally eliminate the need for a labeled molecule.
DETAILED DESCRIPTION OF THE INVENTION
[0048] The present invention relates to methods and compositions for diagnosis, differential diagnosis, risk stratification, monitoring, classifying and determination of treatment regimens in subjects suffering or at risk of suffering from injury to renal function, reduced renal function and/or acute renal failure through measurement of one or more kidney injury markers. In various embodiments, a measured concentration of one or more markers selected from the group consisting of soluble p-selectin, protein NOV homolog, soluble epidermal growth factor receptor, netrin-4, haptoglobin, heat shock protein beta-1, alpha- 1 -antitrypsin, leukocyte elastase, soluble tumor necrosis factor receptor superfamily member 6, soluble tumor necrosis factor ligand superfamily member 6, soluble intercellular adhesion molecule 2, caspase-3 (and most preferably active caspase-3), and soluble platelet endothelial cell adhesion molecule, or one or more markers related thereto, are correlated to the renal status of the subject.
[0049] For purposes of this document, the following definitions apply:
As used herein, an "injury to renal function" is an abrupt (within 14 days, preferably within 7 days, more preferably within 72 hours, and still more preferably within 48 hours) measurable reduction in a measure of renal function. Such an injury may be identified, for example, by a decrease in glomerular filtration rate or estimated GFR, a reduction in urine output, an increase in serum creatinine, an increase in serum cystatin C, a requirement for renal replacement therapy, etc. "Improvement in Renal Function" is an abrupt (within 14 days, preferably within 7 days, more preferably within 72 hours, and still more preferably within 48 hours) measurable increase in a measure of renal function. Preferred methods for measuring and/or estimating GFR are described hereinafter.
As used herein, "reduced renal function" is an abrupt (within 14 days, preferably within 7 days, more preferably within 72 hours, and still more preferably within 48 hours) reduction in kidney function identified by an absolute increase in serum creatinine of greater than or equal to 0.1 mg/dL (> 8.8 μmol/L), a percentage increase in serum creatinine of greater than or equal to 20% (1.2-fold from baseline), or a reduction in urine output (documented oliguria of less than 0. 5 ml/kg per hour).
As used herein, "acute renal failure" or "ARF" is an abrupt (within 14 days, preferably within 7 days, more preferably within 72 hours, and still more preferably within 48 hours) reduction in kidney function identified by an absolute increase in serum creatinine of greater than or equal to 0.3 mg/dl (> 26.4 μmol/1), a percentage increase in serum creatinine of greater than or equal to 50% (1. 5-fold from baseline), or a reduction in urine output (documented oliguria of less than 0.5 ml/kg per hour for at least 6 hours). This term is synonymous with "acute kidney injury" or "AKI."
[0050] In this regard, the skilled artisan will understand that the signals obtained from an immunoassay are a direct result of complexes formed between one or more antibodies and the target biomolecule (i.e., the analyte) and polypeptides containing the necessary epitope(s) to which the antibodies bind. While such assays may detect the full length biomarker and the assay result be expressed as a concentration of a biomarker of interest, the signal from the assay is actually a result of all such "immunoreactive" polypeptides present in the sample. Expression of biomarkers may also be determined by means other than immunoassays, including protein measurements (such as dot blots, western blots, chromatographic methods, mass spectrometry, etc.) and nucleic acid measurements (mRNA quatitation). This list is not meant to be limiting.
[0051] As used herein, the term "p-selectin" refers to one or more polypeptides present in a biological sample that are derived from the p-selectin precursor (Swiss-Prot P16109 (SEQ ID NO: I)).
10 20 30 40 50 60
MANCQIAILY QRFQRVVFGI SQLLCFSALI SELTNQKEVA AWTYHYSTKA YSWNISRKYC
70 80 90 100 110 120
QNRYTDLVAI QNKNEIDYLN KVLPYYSSYY WIGIRKNNKT WTWVGTKKAL TNEAENWADN
130 140 150 160 170 180
EPNNKRNNED CVEIYIKSPS APGKWNDEHC LKKKHALCYT ASCQDMSCSK QGECLETIGN
190 200 210 220 230 240
YTCSCYPGFY GPECEYVREC GELELPQHVL MNCSHPLGNF SFNSQCSFHC TDGYQVNGPS
250 260 270 280 290 300
KLECLASGIW TNKPPQCLAA QCPPLKIPER GNMICLHSAK AFQHQSSCSF SCEEGFALVG
310 320 330 340 350 360
PEVVQCTASG VWTAPAPVCK AVQCQHLEAP SEGTMDCVHP LTAFAYGSSC KFECQPGYRV
370 380 390 400 410 420
RGLDMLRCID SGHWSAPLPT CEAISCEPLE SPVHGSMDCS PSLRAFQYDT NCSFRCAEGF
430 440 450 460 470 480
MLRGADIVRC DNLGQWTAPA PVCQALQCQD LPVPNEARVN CSHPFGAFRY QSVCSFTCNE
490 500 510 520 530 540
GLLLVGASVL QCLATGNWNS VPPECQAIPC TPLLSPQNGT MTCVQPLGSS SYKSTCQFIC
550 560 570 580 590 600 DEGYSLSGPE RLDCTRSGRW TDSPPMCEAI KCPELFAPEQ GSLDCSDTRG EFNVGSTCHF
610 620 630 640 650 660
SCNNGFKLEG PNNVECTTSG RWSATPPTCK GIASLPTPGL QCPALTTPGQ GTMYCRHHPG
670 680 690 700 710 720
TFGFNTTCYF GCNAGFTLIG DSTLSCRPSG QWTAVTPACR AVKCSELHVN KPIAMNCSNL
730 740 750 760 770 780
WGNFSYGSIC SFHCLEGQLL NGSAQTACQE NGHWSTTVPT CQAGPLTIQE ALTYFGGAVA
790 800 810 820 830
STIGLIMGGT LLALLRKRFR QKDDGKCPLN PHSHLGTYGV FTNAAFDPSP
[0052] Most preferably, the p-selectin assay detects one or more soluble forms of p- selectin. P-selectin is a single-pass type I membrane protein having a large extracellular domain, most or all of which is present in soluble forms of p-selectin generated either through alternative splicing event which deletes all or a portion of the transmembrane domain, or by proteolysis of the membrane-bound form. In the case of an immunoassay, one or more antibodies that bind to epitopes within this extracellular domain may be used to detect these soluble form(s). The following domains have been identified in p-selectin:
Residues Length Domain ID
1-41 41 signal sequence
42-830 789 p-selectin
42-771 730 extracellular
772-795 24 transmembrane
796-830 35 cytoplasmic
[0053] As used herein, the term "protein NOV homolog" refers to one or more polypeptides present in a biological sample that are derived from the protein NOV homolog precursor (Swiss-Prot P48745 (SEQ ID NO: 2)).
10 20 30 40 50 60
MQSVQSTSFC LRKQCLCLTF LLLHLLGQVA ATQRCPPQCP GRCPATPPTC APGVRAVLDG
70 80 90 100 110 120
CSCCLVCARQ RGESCSDLEP CDESSGLYCD RSADPSNQTG ICTAVEGDNC VFDGVIYRSG 130 140 150 160 170 180
EKFQPSCKFQ CTCRDGQIGC VPRCQLDVLL PEPNCPAPRK VEVPGECCEK WICGPDEEDS
190 200 210 220 230 240
LGGLTLAAYR PEATLGVEVS DSSVNCIEQT TEWTACSKSC GMGFSTRVTN RNRQCEMLKQ
250 260 270 280 290 300
TRLCMVRPCE QEPEQPTDKK GKKCLRTKKS LKAIHLQFKN CTSLHTYKPR FCGVCSDGRC
310 320 330 340 350
CTPHNTKTIQ AEFQCSPGQI VKKPVMVIGT CTCHTNCPKN NEAFLQELEL KTTRGKM
[0054] The following domains have been identified in protein NOV homolog:
Residues Length Domain ID
1-31 31 signal sequence
32-357 326 protein NOV homolog
[0055] As used herein, the term "epidermal growth factor receptor" refers to one or more polypeptides present in a biological sample that are derived from the epidermal growth factor receptor precursor (Swiss-Prot P00533 (SEQ ID NO: 3)).
10 20 30 40 50 60
MRPSGTAGAA LLALLAALCP ASRALEEKKV CQGTSNKLTQ LGTFEDHFLS LQRMFNNCEV
70 80 90 100 110 120
VLGNLEITYV QRNYDLSFLK TIQEVAGYVL IALNTVERIP LENLQIIRGN MYYENSYALA
130 140 150 160 170 180
VLSNYDANKT GLKELPMRNL QEILHGAVRF SNNPALCNVE SIQWRDIVSS DFLSNMSMDF
190 200 210 220 230 240
QNHLGSCQKC DPSCPNGSCW GAGEENCQKL TKIICAQQCS GRCRGKSPSD CCHNQCAAGC
250 260 270 280 290 300
TGPRESDCLV CRKFRDEATC KDTCPPLMLY NPTTYQMDVN PEGKYSFGAT CVKKCPRNYV
310 320 330 340 350 360
VTDHGSCVRA CGADSYEMEE DGVRKCKKCE GPCRKVCNGI GIGEFKDSLS INATNIKHFK
370 380 390 400 410 420 NCTSISGDLH ILPVAFRGDS FTHTPPLDPQ ELDILKTVKE ITGFLLIQAW PENRTDLHAF
430 440 450 460 470 480
ENLEIIRGRT KQHGQFSLAV VSLNITSLGL RSLKEISDGD VIISGNKNLC YANTINWKKL
490 500 510 520 530 540
FGTSGQKTKI ISNRGENSCK ATGQVCHALC SPEGCWGPEP RDCVSCRNVS RGRECVDKCN
550 560 570 580 590 600
LLEGEPREFV ENSECIQCHP ECLPQAMNIT CTGRGPDNCI QCAHYIDGPH CVKTCPAGVM
610 620 630 640 650 660
GENNTLVWKY ADAGHVCHLC HPNCTYGCTG PGLEGCPTNG PKIPSIATGM VGALLLLLVV
670 680 690 700 710 720
ALGIGLFMRR RHIVRKRTLR RLLQERELVE PLTPSGEAPN QALLRILKET EFKKIKVLGS
730 740 750 760 770 780
GAFGTVYKGL WIPEGEKVKI PVAIKELREA TSPKANKEIL DEAYVMASVD NPHVCRLLGI
790 800 810 820 830 840
CLTSTVQLIT QLMPFGCLLD YVREHKDNIG SQYLLNWCVQ IAKGMNYLED RRLVHRDLAA
850 860 870 880 890 900
RNVLVKTPQH VKITDFGLAK LLGAEEKEYH AEGGKVPIKW MALESILHRI YTHQSDVWSY
910 920 930 940 950 960
GVTVWELMTF GSKPYDGIPA SEISSILEKG ERLPQPPICT IDVYMIMVKC WMIDADSRPK
970 980 990 1000 1010 1020
FRELIIEFSK MARDPQRYLV IQGDERMHLP SPTDSNFYRA LMDEEDMDDV VDADEYLIPQ
1030 1040 1050 1060 1070 1080
QGFFSSPSTS RTPLLSSLSA TSNNSTVACI DRNGLQSCPI KEDSFLQRYS SDPTGALTED
1090 1100 1110 1120 1130 1140
SIDDTFLPVP EYINQSVPKR PAGSVQNPVY HNQPLNPAPS RDPHYQDPHS TAVGNPEYLN
1150 1160 1170 1180 1190 1200
TVQPTCVNST FDSPAHWAQK GSHQISLDNP DYQQDFFPKE AKPNGIFKGS TAENAEYLRV
1210 APQSSEFIGA
[0056] Most preferably, the epidermal growth factor receptor assay detects one or more soluble forms of epidermal growth factor receptor. Epidermal growth factor receptor is a single-pass type I membrane protein having a large extracellular domain, most or all of which is present in soluble forms of epidermal growth factor receptor generated either through alternative splicing event which deletes all or a portion of the transmembrane domain, or by proteolysis of the membrane-bound form. In the case of an immunoassay, one or more antibodies that bind to epitopes within this extracellular domain may be used to detect these soluble form(s). The following domains have been identified in epidermal growth factor receptor:
Residues Length Domain ID
1-24 24 signal sequence
25-1210 1186 epidermal growth factor receptor
25-645 621 extracellular
646-668 23 transmembrane
669-1210 542 cytoplasmic
[0057] As used herein, the term "netrin-4" refers to one or more polypeptides present in a biological sample that are derived from the netrin-4 precursor (Swiss-Prot Q9HB63 (SEQ ID NO: 4)).
10 20 30 40 50 60
MGSCARLLLL WGCTVVAAGL SGVAGVSSRC EKACNPRMGN LALGRKLWAD TTCGQNATEL
70 80 90 100 110 120
YCFYSENTDL TCRQPKCDKC NAAYPHLAHL PSAMADSSFR FPRTWWQSAE DVHREKIQLD
130 140 150 160 170 180
LEAEFYFTHL IVMFKSPRPA AMVLDRSQDF GKTWKPYKYF ATNCSATFGL EDDVVKKGAI
190 200 210 220 230 240
CTSKYSSPFP CTGGEVIFKA LSPPYDTENP YSAKVQEQLK ITNLRVQLLK RQSCPCQRND
250 260 270 280 290 300
LNEEPQHFTH YAIYDFIVKG SCFCNGHADQ CIPVHGFRPV KAPGTFHMVH GKCMCKHNTA 310 320 330 340 350 360
GSHCQHCAPL YNDRPWEAAD GKTGAPNECR TCKCNGHADT CHFDVNVWEA SGNRSGGVCD
370 380 390 400 410 420
DCQHNTEGQY CQRCKPGFYR DLRRPFSAPD ACKPCSCHPV GSAVLPANSV TFCDPSNGDC
430 440 450 460 470 480
PCKPGVAGRR CDRCMVGYWG FGDYGCRPCD CAGSCDPITG DCISSHTDID WYHEVPDFRP
490 500 510 520 530 540
VHNKSEPAWE WEDAQGFSAL LHSGKCECKE QTLGNAKAFC GMKYSYVLKI KILSAHDKGT
550 560 570 580 590 600
HVEVNVKIKK VLKSTKLKIF RGKRTLYPES WTDRGCTCPI LNPGLEYLVA GHEDIRTGKL
610 620 IVNMKSFVQH WKPSLGRKVM DILKRECK
[0058] The following domains have been identified in netrin-4:
Residues Length Domain ID
1-18 18 initiator methionine
19-628 610 netrin-4
[0059] As used herein, the term "haptoglobin" refers to one or more polypeptides present in a biological sample that are derived from the haptoglobin precursor (Swiss-Prot P00738 (SEQ ID NO: 5)).
10 20 30 40 50 60
MSALGAVIAL LLWGQLFAVD SGNDVTDIAD DGCPKPPEIA HGYVEHSVRY QCKNYYKLRT
70 80 90 100 110 120
EGDGVYTLND KKQWINKAVG DKLPECEADD GCPKPPEIAH GYVEHSVRYQ CKNYYKLRTE
130 140 150 160 170 180
GDGVYTLNNE KQWINKAVGD KLPECEAVCG KPKNPANPVQ RILGGHLDAK GSFPWQAKMV
190 200 210 220 230 240
SHHNLTTGAT LINEQWLLTT AKNLFLNHSE NATAKDIAPT LTLYVGKKQL VEIEKVVLHP
250 260 270 280 290 300 NYSQVDIGLI KLKQKVSVNE RVMPICLPSK DYAEVGRVGY VSGWGRNANF KFTDHLKYVM 310 320 330 340 350 360
LPVADQDQCI RHYEGSTVPE KKTPKSPVGV QPILNEHTFC AGMSKYQEDT CYGDAGSAFA 370 380 390 400
VHDLEEDTWY ATGILSFDKS CAVAEYGVYV KVTSIQDWVQ KTIAEN
[0060] The following domains have been identified in haptoglobin:
Residues Length Domain ID
1-18 18 signal sequence
19-406 388 haptoglobin
19-160 142 haptoglobin alpha chain
162-406 245 haptoglobin beta chain
[0061] As used herein, the term "alpha- 1 -antitrypsin" refers to one or more polypeptides present in a biological sample that are derived from the alpha- 1- antitrypsin precursor (Swiss- Prot P01009 (SEQ ID NO: 6)).
10 20 30 40 50 60
MPSSVSWGIL LLAGLCCLVP VSLAEDPQGD AAQKTDTSHH DQDHPTFNKI TPNLAEFAFS
70 80 90 100 110 120
LYRQLAHQSN STNIFFSPVS IATAFAMLSL GTKADTHDEI LEGLNFNLTE IPEAQIHEGF
130 140 150 160 170 180
QELLRTLNQP DSQLQLTTGN GLFLSEGLKL VDKFLEDVKK LYHSEAFTVN FGDTEEAKKQ
190 200 210 220 230 240
INDYVEKGTQ GKIVDLVKEL DRDTVFALVN YIFFKGKWER PFEVKDTEEE DFHVDQVTTV
250 260 270 280 290 300
KVPMMKRLGM FNIQHCKKLS SWVLLMKYLG NATAIFFLPD EGKLQHLENE LTHDIITKFL
310 320 330 340 350 360
ENEDRRSASL HLPKLSITGT YDLKSVLGQL GITKVFSNGA DLSGVTEEAP LKLSKAVHKA
370 380 390 400 410
VLTIDEKGTE AAGAMFLEAI PMSIPPEVKF NKPFVFLMIE QNTKSPLFMG KVVNPTQK [0062] The following domains have been identified in alpha- 1 -antitrypsin:
Residues Length Domain ID
1-24 24 signal sequence
25-418 394 alpha- 1 -antitrypsin
[0063] As used herein, the term "leukocyte elastase" refers to one or more polypeptides present in a biological sample that are derived from the leukocyte elastase precursor (Swiss- Prot P08246 (SEQ ID NO: 7)).
10 20 30 40 50 60
MTLGRRLACL FLACVLPALL LGGTALASEI VGGRRARPHA WPFMVSLQLR GGHFCGATLI
70 80 90 100 110 120
APNFVMSAAH CVANVNVRAV RVVLGAHNLS RREPTRQVFA VQRIFENGYD PVNLLNDIVI
130 140 150 160 170 180
LQLNGSATIN ANVQVAQLPA QGRRLGNGVQ CLAMGWGLLG RNRGIASVLQ ELNVTVVTSL
190 200 210 220 230 240
CRRSNVCTLV RGRQAGVCFG DSGSPLVCNG LIHGIASFVR GGCASGLYPD AFAPVAQFVN
250 260
WIDSIIQRSE DNPCPHPRDP DPASRTH
[0064] The following domains have been identified in leukocyte elastase:
Residues Length Domain ID
1-27 315 signal sequence
28-29 2 pro-peptide
30-267 238 leukocyte elastase
[0065] As used herein, the term "tumor necrosis factor receptor superfamily member 6" refers to one or more polypeptides present in a biological sample that are derived from the tumor necrosis factor receptor superfamily member 6 precursor (Swiss-Prot P25445 (SEQ ID NO: 8)).
10 20 30 40 50 60 MLGIWTLLPL VLTSVARLSS KSVNAQVTDI NSKGLELRKT VTTVETQNLE GLHHDGQFCH 70 80 90 100 110 120 KPCPPGERKA RDCTVNGDEP DCVPCQEGKE YTDKAHFSSK CRRCRLCDEG HGLEVEINCT
130 140 150 160 170 180 RTQNTKCRCK PNFFCNSTVC EHCDPCTKCE HGIIKECTLT SNTKCKEEGS RSNLGWLCLL
190 200 210 220 230 240 LLPIPLIVWV KRKEVQKTCR KHRKENQGSH ESPTLNPETV AINLSDVDLS KYITTIAGVM
250 260 270 280 290 300 TLSQVKGFVR KNGVNEAKID EIKNDNVQDT AEQKVQLLRN WHQLHGKKEA YDTLIKDLKK
310 320 330 ANLCTLAEKI QTIILKDITS DSENSNFRNE IQSLV
[0066] Most preferably, the tumor necrosis factor receptor superfamily member 6 assay detects one or more soluble forms of tumor necrosis factor receptor superfamily member 6. Tumor necrosis factor receptor superfamily member 6 is a single-pass type I membrane protein having a large extracellular domain, most or all of which is present in soluble forms of tumor necrosis factor receptor superfamily member 6 generated either through alternative splicing event which deletes all or a portion of the transmembrane domain, or by proteolysis of the membrane-bound form. In the case of an immunoassay, one or more antibodies that bind to epitopes within this extracellular domain may be used to detect these soluble form(s). The following domains have been identified in tumor necrosis factor receptor superfamily member 6:
Residues Length Domain ID
1-25 25 signal sequence
26-335 310 tumor necrosis factor receptor superfamily member 6
26-173 148 extracellular
174-190 17 transmembrane
191-335 145 cytoplasmic
[0067] As used herein, the term "tumor necrosis factor ligand superfamily member 6" refers to one or more polypeptides present in a biological sample that are derived from the tumor necrosis factor ligand superfamily member 6 precursor (Swiss-Prot P48023 (SEQ ID NO: 9)). 10 20 30 40 50 60
MQQPFNYPYP QIYWVDSSAS SPWAPPGTVL PCPTSVPRRP GQRRPPPPPP PPPLPPPPPP
70 80 90 100 110 120
PPLPPLPLPP LKKRGNHSTG LCLLVMFFMV LVALVGLGLG MFQLFHLQKE LAELRESTSQ
130 140 150 160 170 180
MHTASSLEKQ IGHPSPPPEK KELRKVAHLT GKSNSRSMPL EWEDTYGIVL LSGVKYKKGG
190 200 210 220 230 240
LVINETGLYF VYSKVYFRGQ SCNNLPLSHK VYMRNSKYPQ DLVMMEGKMM SYCTTGQMWA
250 260 270 280
RSSYLGAVFN LTSADHLYVN VSELSLVNFE ESQTFFGLYK L
[0068] Most preferably, the tumor necrosis factor ligand superfamily member 6 assay detects one or more soluble forms of tumor necrosis factor ligand superfamily member 6. Tumor necrosis factor ligand superfamily member 6 is a single-pass type II membrane protein having a large extracellular domain, most or all of which is present in soluble forms of tumor necrosis factor ligand superfamily member 6 generated either through alternative splicing event which deletes all or a portion of the transmembrane domain, or by proteolysis of the membrane-bound form. In the case of an immunoassay, one or more antibodies that bind to epitopes within this extracellular domain may be used to detect these soluble form(s). The following domains have been identified in tumor necrosis factor ligand superfamily member 6:
Residues Length Domain ID
1-281 281 necrosis factor ligand superfamily member 6, membrane bound form
130-281 152 tumor necrosis factor ligand superfamily member 6, soluble form
1-180 180 cytoplasmic
81 - 102 22 membrane anchor signal
103-281 179 extracellular [0069] As used herein, the term "intercellular adhesion molecule 2" refers to one or more polypeptides present in a biological sample that are derived from the intercellular adhesion molecule 2 precursor (Swiss-Prot P13598 (SEQ ID NO: 10)).
10 20 30 40 50 60
MSSFGYRTLT VALFTLICCP GSDEKVFEVH VRPKKLAVEP KGSLEVNCST TCNQPEVGGL
70 80 90 100 110 120
ETSLDKILLD EQAQWKHYLV SNISHDTVLQ CHFTCSGKQE SMNSNVSVYQ PPRQVILTLQ
130 140 150 160 170 180
PTLVAVGKSF TIECRVPTVE PLDSLTLFLF RGNETLHYET FGKAAPAPQE ATATFNSTAD
190 200 210 220 230 240
REDGHRNFSC LAVLDLMSRG GNIFHKHSAP KMLEIYEPVS DSQMVIIVTV VSVLLSLFVT
250 260 270
SVLLCFIFGQ HLRQQRMGTY GVRAAWRRLP QAFRP
[0070] Most preferably, the intercellular adhesion molecule 2 assay detects one or more soluble forms of intercellular adhesion molecule 2. Intercellular adhesion molecule 2 is a single-pass type I membrane protein having a large extracellular domain, most or all of which is present in soluble forms of intercellular adhesion molecule 2 generated either through alternative splicing event which deletes all or a portion of the transmembrane domain, or by proteolysis of the membrane-bound form. In the case of an immunoassay, one or more antibodies that bind to epitopes within this extracellular domain may be used to detect these soluble form(s). The following domains have been identified in intercellular adhesion molecule 2:
Residues Length Domain ID
1-21 21 signal sequence
22-275 254 intercellular adhesion molecule 2
22-223 202 extracellular
224-248 25 transmembrane
249-275 27 cytoplasmic [0071] As used herein, the term "caspase-3" refers to one or more polypeptides present in a biological sample that are derived from the caspase-3 precursor (Swiss-Prot P42574 (SEQ ID NO: H)).
10 20 30 40 50 60
MENTENSVDS KSIKNLEPKI IHGSESMDSG ISLDNSYKMD YPEMGLCIII NNKNFHKSTG
70 80 90 100 110 120
MTSRSGTDVD AANLRETFRN LKYEVRNKND LTREEIVELM RDVSKEDHSK RSSFVCVLLS
130 140 150 160 170 180
HGEEGIIFGT NGPVDLKKIT NFFRGDRCRS LTGKPKLFII QACRGTELDC GIETDSGVDD
190 200 210 220 230 240
DMACHKIPVE ADFLYAYSTA PGYYSWRNSK DGSWFIQSLC AMLKQYADKL EFMHILTRVN
250 260 270
RKVATEFESF SFDATFHAKK QIPCIVSMLT KELYFYH
[0072] The following domains have been identified in caspase-3:
Residues Length Domain ID
1-9 9 Propeptide
10-28 19 propeptide
29-175 147 caspase-3 pl7 subunit
176-277 102 caspase-3 pl2 subunit
[0073] Suitable assays may recognize only the pl7 subunit of caspase-3, may recognize only the pl2 subunit of caspase-3 (24 kDa) but not the full length caspase-3, may recognize only full length caspase-3, or may recognize one subunit and the full length full length caspase-3. In this regard, the skilled artisan will understand that the signals obtained from an immunoassay are a direct result of complexes formed between one or more antibodies and the target biomolecule (i.e., the analyte) and polypeptides containing the necessary epitope(s) to which the antibodies bind. While such assays may detect the full length caspase-3 molecule and the assay result be expressed as a concentration of caspase-3, the signal from the assay is actually a result of all such "immunoreactive" polypeptides present in the sample. [0074] As used herein, the term "platelet endothelial cell adhesion molecule" refers to one or more polypeptides present in a biological sample that are derived from the platelet endothelial cell adhesion molecule precursor (Swiss-Prot P16284 (SEQ ID NO: 12)).
10 20 30 40 50 60
MQPRWAQGAT MWLGVLLTLL LCSSLEGQEN SFTINSVDMK SLPDWTVQNG KNLTLQCFAD
70 80 90 100 110 120
VSTTSHVKPQ HQMLFYKDDV LFYNISSMKS TESYFIPEVR IYDSGTYKCT VIVNNKEKTT
130 140 150 160 170 180
AEYQLLVEGV PSPRVTLDKK EAIQGGIVRV NCSVPEEKAP IHFTIEKLEL NEKMVKLKRE
190 200 210 220 230 240
KNSRDQNFVI LEFPVEEQDR VLSFRCQARI ISGIHMQTSE STKSELVTVT ESFSTPKFHI
250 260 270 280 290 300
SPTGMIMEGA QLHIKCTIQV THLAQEFPEI I IQKDKAIVA HNRHGNKAVY SVMAMVEHSG
310 320 330 340 350 360
NYTCKVESSR ISKVSSIVVN ITELFSKPEL ESSFTHLDQG ERLNLSCSIP GAPPANFTIQ
370 380 390 400 410 420
KEDTIVSQTQ DFTKIASKSD SGTYICTAGI DKVVKKSNTV QIVVCEMLSQ PRISYDAQFE
430 440 450 460 470 480
VIKGQTIEVR CESISGTLPI SYQLLKTSKV LENSTKNSND PAVFKDNPTE DVEYQCVADN
490 500 510 520 530 540
CHSHAKMLSE VLRVKVIAPV DEVQISILSS KVVESGEDIV LQCAVNEGSG PITYKFYREK
550 560 570 580 590 600
EGKPFYQMTS NATQAFWTKQ KASKEQEGEY YCTAFNRANH ASSVPRSKIL TVRVILAPWK
610 620 630 640 650 660
KGLIAVVIIG VIIALLIIAA KCYFLRKAKA KQMPVEMSRP AVPLLNSNNE KMSDPNMEAN
670 680 690 700 710 720
SHYGHNDDVR NHAMKPINDN KEPLNSDVQY TEVQVSSAES HKDLGKKDTE TVYSEVRKAV
730 PDAVESRYSR TEGSLDGT [0075] Most preferably, the platelet endothelial cell adhesion molecule assay detects one or more soluble forms of platelet endothelial cell adhesion molecule. Platelet endothelial cell adhesion molecule is a single-pass type I membrane protein having a large extracellular domain, most or all of which is present in soluble forms of platelet endothelial cell adhesion molecule generated either through alternative splicing event which deletes all or a portion of the transmembrane domain, or by proteolysis of the membrane-bound form. In the case of an immunoassay, one or more antibodies that bind to epitopes within this extracellular domain may be used to detect these soluble form(s). The following domains have been identified in platelet endothelial cell adhesion molecule:
Residues Length Domain ID
1-27 27 signal sequence
28-738 711 platelet endothelial cell adhesion molecule
28-601 574 extracellular
602-620 19 transmembrane
621-738 118 cytoplasmic
[0076] As used herein, the term "heat shock protein beta-1" refers to one or more polypeptides present in a biological sample that are derived from the heat shock protein beta- 1 precursor (Swiss-Prot P04792 (SEQ ID NO: 13)).
10 20 30 40 50 60
MTERRVPFSL LRGPSWDPFR DWYPHSRLFD QAFGLPRLPE EWSQWLGGSS WPGYVRPLPP
70 80 90 100 110 120
AAIESPAVAA PAYSRALSRQ LSSGVSEIRH TADRWRVSLD VNHFAPDELT VKTKDGVVEI
130 140 150 160 170 180
TGKHEERQDE HGYISRCFTR KYTLPPGVDP TQVSSSLSPE GTLTVEAPMP KLATQSNEIT
190 200
IPVTFESRAQ LGGPEAAKSD ETAAK
[0077] As used herein, the term "epidermal growth factor receptor" refers to one or more polypeptides present in a biological sample that are derived from the epidermal growth factor receptor precursor (Swiss-Prot P00533 (SEQ ID NO: 14)). 10 20 30 40 50 60
MRPSGTAGAA LLALLAALCP ASRALEEKKV CQGTSNKLTQ LGTFEDHFLS LQRMFNNCEV
70 80 90 100 110 120
VLGNLEITYV QRNYDLSFLK TIQEVAGYVL IALNTVERIP LENLQIIRGN MYYENSYALA
130 140 150 160 170 180
VLSNYDANKT GLKELPMRNL QEILHGAVRF SNNPALCNVE SIQWRDIVSS DFLSNMSMDF
190 200 210 220 230 240
QNHLGSCQKC DPSCPNGSCW GAGEENCQKL TKIICAQQCS GRCRGKSPSD CCHNQCAAGC
250 260 270 280 290 300
TGPRESDCLV CRKFRDEATC KDTCPPLMLY NPTTYQMDVN PEGKYSFGAT CVKKCPRNYV
310 320 330 340 350 360
VTDHGSCVRA CGADSYEMEE DGVRKCKKCE GPCRKVCNGI GIGEFKDSLS INATNIKHFK
370 380 390 400 410 420
NCTSISGDLH ILPVAFRGDS FTHTPPLDPQ ELDILKTVKE ITGFLLIQAW PENRTDLHAF
430 440 450 460 470 480
ENLEIIRGRT KQHGQFSLAV VSLNITSLGL RSLKEISDGD VIISGNKNLC YANTINWKKL
490 500 510 520 530 540
FGTSGQKTKI ISNRGENSCK ATGQVCHALC SPEGCWGPEP RDCVSCRNVS RGRECVDKCN
550 560 570 580 590 600
LLEGEPREFV ENSECIQCHP ECLPQAMNIT CTGRGPDNCI QCAHYIDGPH CVKTCPAGVM
610 620 630 640 650 660
GENNTLVWKY ADAGHVCHLC HPNCTYGCTG PGLEGCPTNG PKIPSIATGM VGALLLLLVV
670 680 690 700 710 720
ALGIGLFMRR RHIVRKRTLR RLLQERELVE PLTPSGEAPN QALLRILKET EFKKIKVLGS
730 740 750 760 770 780
GAFGTVYKGL WIPEGEKVKI PVAIKELREA TSPKANKEIL DEAYVMASVD NPHVCRLLGI
790 800 810 820 830 840
CLTSTVQLIT QLMPFGCLLD YVREHKDNIG SQYLLNWCVQ IAKGMNYLED RRLVHRDLAA 850 860 870 880 890 900
RNVLVKTPQH VKITDFGLAK LLGAEEKEYH AEGGKVPIKW MALESILHRI YTHQSDVWSY
910 920 930 940 950 960
GVTVWELMTF GSKPYDGIPA SEISSILEKG ERLPQPPICT IDVYMIMVKC WMIDADSRPK
970 980 990 1000 1010 1020
FRELIIEFSK MARDPQRYLV IQGDERMHLP SPTDSNFYRA LMDEEDMDDV VDADEYLIPQ
1030 1040 1050 1060 1070 1080
QGFFSSPSTS RTPLLSSLSA TSNNSTVACI DRNGLQSCPI KEDSFLQRYS SDPTGALTED
1090 1100 1110 1120 1130 1140
SIDDTFLPVP EYINQSVPKR PAGSVQNPVY HNQPLNPAPS RDPHYQDPHS TAVGNPEYLN
1150 1160 1170 1180 1190 1200
TVQPTCVNST FDSPAHWAQK GSHQISLDNP DYQQDFFPKE AKPNGIFKGS TAENAEYLRV
1210 APQSSEFIGA
[0078] Most preferably, the epidermal growth factor receptor assay detects one or more soluble forms of epidermal growth factor receptor. Epidermal growth factor receptor is a single-pass type I membrane protein having a large extracellular domain, most or all of which is present in soluble forms of epidermal growth factor receptor generated either through alternative splicing event which deletes all or a portion of the transmembrane domain, or by proteolysis of the membrane-bound form. In the case of an immunoassay, one or more antibodies that bind to epitopes within this extracellular domain may be used to detect these soluble form(s). The following domains have been identified in epidermal growth factor receptor:
Residues Length Domain ID
1-24 24 signal sequence
25-1210 1186 epidermal growth factor receptor
25-645 621 extracellular
646-668 23 transmembrane
669-1210 542 cytoplasmic [0079] As used herein, the term "relating a signal to the presence or amount" of an analyte reflects this understanding. Assay signals are typically related to the presence or amount of an analyte through the use of a standard curve calculated using known concentrations of the analyte of interest. As the term is used herein, an assay is "configured to detect" an analyte if an assay can generate a detectable signal indicative of the presence or amount of a physiologically relevant concentration of the analyte. Because an antibody epitope is on the order of 8 amino acids, an immunoassay configured to detect a marker of interest will also detect polypeptides related to the marker sequence, so long as those polypeptides contain the epitope(s) necessary to bind to the antibody or antibodies used in the assay. The term "related marker" as used herein with regard to a biomarker such as one of the kidney injury markers described herein refers to one or more fragments, variants, etc., of a particular marker or its biosynthetic parent that may be detected as a surrogate for the marker itself or as independent biomarkers. The term also refers to one or more polypeptides present in a biological sample that are derived from the biomarker precursor complexed to additional species, such as binding proteins, receptors, heparin, lipids, sugars, etc.
[0080] The term "positive going" marker as that term is used herein refer to a marker that is determined to be elevated in subjects suffering from a disease or condition, relative to subjects not suffering from that disease or condition. The term "negative going" marker as that term is used herein refer to a marker that is determined to be reduced in subjects suffering from a disease or condition, relative to subjects not suffering from that disease or condition.
[0081] The term "subject" as used herein refers to a human or non-human organism. Thus, the methods and compositions described herein are applicable to both human and veterinary disease. Further, while a subject is preferably a living organism, the invention described herein may be used in post-mortem analysis as well. Preferred subjects are humans, and most preferably "patients," which as used herein refers to living humans that are receiving medical care for a disease or condition. This includes persons with no defined illness who are being investigated for signs of pathology.
[0082] Preferably, an analyte is measured in a sample. Such a sample may be obtained from a subject, or may be obtained from biological materials intended to be provided to the subject. For example, a sample may be obtained from a kidney being evaluated for possible transplantation into a subject, and an analyte measurement used to evaluate the kidney for preexisting damage. Preferred samples are body fluid samples. [0083] The term "body fluid sample" as used herein refers to a sample of bodily fluid obtained for the purpose of diagnosis, prognosis, classification or evaluation of a subject of interest, such as a patient or transplant donor. In certain embodiments, such a sample may be obtained for the purpose of determining the outcome of an ongoing condition or the effect of a treatment regimen on a condition. Preferred body fluid samples include blood, serum, plasma, cerebrospinal fluid, urine, saliva, sputum, and pleural effusions. In addition, one of skill in the art would realize that certain body fluid samples would be more readily analyzed following a fractionation or purification procedure, for example, separation of whole blood into serum or plasma components.
[0084] The term "diagnosis" as used herein refers to methods by which the skilled artisan can estimate and/or determine the probability ("a likelihood") of whether or not a patient is suffering from a given disease or condition. In the case of the present invention, "diagnosis" includes using the results of an assay, most preferably an immunoassay, for a kidney injury marker of the present invention, optionally together with other clinical characteristics, to arrive at a diagnosis (that is, the occurrence or nonoccurrence) of an acute renal injury or ARF for the subject from which a sample was obtained and assayed. That such a diagnosis is "determined" is not meant to imply that the diagnosis is 100% accurate. Many biomarkers are indicative of multiple conditions. The skilled clinician does not use biomarker results in an informational vacuum, but rather test results are used together with other clinical indicia to arrive at a diagnosis. Thus, a measured biomarker level on one side of a predetermined diagnostic threshold indicates a greater likelihood of the occurrence of disease in the subject relative to a measured level on the other side of the predetermined diagnostic threshold.
[0085] Similarly, a prognostic risk signals a probability ("a likelihood") that a given course or outcome will occur. A level or a change in level of a prognostic indicator, which in turn is associated with an increased probability of morbidity (e.g., worsening renal function, future ARF, or death) is referred to as being "indicative of an increased likelihood" of an adverse outcome in a patient.
[0086] Marker Assays
[0087] In general, immunoassays involve contacting a sample containing or suspected of containing a biomarker of interest with at least one antibody that specifically binds to the biomarker. A signal is then generated indicative of the presence or amount of complexes formed by the binding of polypeptides in the sample to the antibody. The signal is then related to the presence or amount of the biomarker in the sample. Numerous methods and devices are well known to the skilled artisan for the detection and analysis of biomarkers. See, e.g., U.S. Patents 6,143,576; 6,113,855; 6,019,944; 5,985,579; 5,947,124; 5,939,272; 5,922,615; 5,885,527; 5,851,776; 5,824,799; 5,679,526; 5,525,524; and 5,480,792, and The Immunoassay Handbook, David Wild, ed. Stockton Press, New York, 1994, each of which is hereby incorporated by reference in its entirety, including all tables, figures and claims.
[0088] The assay devices and methods known in the art can utilize labeled molecules in various sandwich, competitive, or non-competitive assay formats, to generate a signal that is related to the presence or amount of the biomarker of interest. Suitable assay formats also include chromatographic, mass spectrographic, and protein "blotting" methods. Additionally, certain methods and devices, such as biosensors and optical immunoassays, may be employed to determine the presence or amount of analytes without the need for a labeled molecule. See, e.g., U.S. Patents 5,631,171; and 5,955,377, each of which is hereby incorporated by reference in its entirety, including all tables, figures and claims. One skilled in the art also recognizes that robotic instrumentation including but not limited to Beckman ACCESS®, Abbott AXSYM®, Roche ELECSYS®, Dade Behring STRATUS® systems are among the immunoassay analyzers that are capable of performing immunoassays. But any suitable immunoassay may be utilized, for example, enzyme-linked immunoassays (ELISA), radioimmunoassays (RIAs), competitive binding assays, and the like.
[0089] Antibodies or other polypeptides may be immobilized onto a variety of solid supports for use in assays. Solid phases that may be used to immobilize specific binding members include include those developed and/or used as solid phases in solid phase binding assays. Examples of suitable solid phases include membrane filters, cellulose-based papers, beads (including polymeric, latex and paramagnetic particles), glass, silicon wafers, microparticles, nanoparticles, TentaGels, AgroGels, PEGA gels, SPOCC gels, and multiple- well plates. An assay strip could be prepared by coating the antibody or a plurality of antibodies in an array on solid support. This strip could then be dipped into the test sample and then processed quickly through washes and detection steps to generate a measurable signal, such as a colored spot. Antibodies or other polypeptides may be bound to specific zones of assay devices either by conjugating directly to an assay device surface, or by indirect binding. In an example of the later case, antibodies or other polypeptides may be immobilized on particles or other solid supports, and that solid support immobilized to the device surface.
[0090] Biological assays require methods for detection, and one of the most common methods for quantitation of results is to conjugate a detectable label to a protein or nucleic acid that has affinity for one of the components in the biological system being studied. Detectable labels may include molecules that are themselves detectable (e.g., fluorescent moieties, electrochemical labels, metal chelates, etc.) as well as molecules that may be indirectly detected by production of a detectable reaction product (e.g., enzymes such as horseradish peroxidase, alkaline phosphatase, etc.) or by a specific binding molecule which itself may be detectable (e.g., biotin, digoxigenin, maltose, oligohistidine, 2,4-dintrobenzene, phenylarsenate, ssDNA, dsDNA, etc.).
[0091] Preparation of solid phases and detectable label conjugates often comprise the use of chemical cross-linkers. Cross-linking reagents contain at least two reactive groups, and are divided generally into homofunctional cross-linkers (containing identical reactive groups) and heterofunctional cross-linkers (containing non-identical reactive groups). Homobifunctional cross-linkers that couple through amines, sulfhydryls or react non- specifically are available from many commercial sources. Maleimides, alkyl and aryl halides, alpha-haloacyls and pyridyl disulfides are thiol reactive groups. Maleimides, alkyl and aryl halides, and alpha-haloacyls react with sulfhydryls to form thiol ether bonds, while pyridyl disulfides react with sulfhydryls to produce mixed disulfides. The pyridyl disulfide product is cleavable. Imidoesters are also very useful for protein-protein cross-links. A variety of heterobifunctional cross -linkers, each combining different attributes for successful conjugation, are commercially available.
[0092] In certain aspects, the present invention provides kits for the analysis of the described kidney injury markers. The kit comprises reagents for the analysis of at least one test sample which comprise at least one antibody that a kidney injury marker. The kit can also include devices and instructions for performing one or more of the diagnostic and/or prognostic correlations described herein. Preferred kits will comprise an antibody pair for performing a sandwich assay, or a labeled species for performing a competitive assay, for the analyte. Preferably, an antibody pair comprises a first antibody conjugated to a solid phase and a second antibody conjugated to a detectable label, wherein each of the first and second antibodies that bind a kidney injury marker. Most preferably each of the antibodies are monoclonal antibodies. The instructions for use of the kit and performing the correlations can be in the form of labeling, which refers to any written or recorded material that is attached to, or otherwise accompanies a kit at any time during its manufacture, transport, sale or use. For example, the term labeling encompasses advertising leaflets and brochures, packaging materials, instructions, audio or video cassettes, computer discs, as well as writing imprinted directly on kits.
[0093] Antibodies
[0094] The term "antibody" as used herein refers to a peptide or polypeptide derived from, modeled after or substantially encoded by an immunoglobulin gene or immunoglobulin genes, or fragments thereof, capable of specifically binding an antigen or epitope. See, e.g. Fundamental Immunology, 3rd Edition, W.E. Paul, ed., Raven Press, N.Y. (1993); Wilson (1994; J. Immunol. Methods 175:267-273; Yarmush (1992) J. Biochem. Biophys. Methods 25:85-97. The term antibody includes antigen-binding portions, i.e., "antigen binding sites," (e.g., fragments, subsequences, complementarity determining regions (CDRs)) that retain capacity to bind antigen, including (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CHl domains; (ii) a F(ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VH and CHl domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (v) a dAb fragment (Ward et al., (1989) Nature 341:544-546), which consists of a VH domain; and (vi) an isolated complementarity determining region (CDR). Single chain antibodies are also included by reference in the term "antibody."
[0095] Antibodies used in the immunoassays described herein preferably specifically bind to a kidney injury marker of the present invention. The term "specifically binds" is not intended to indicate that an antibody binds exclusively to its intended target since, as noted above, an antibody binds to any polypeptide displaying the epitope(s) to which the antibody binds. Rather, an antibody "specifically binds" if its affinity for its intended target is about 5- fold greater when compared to its affinity for a non-target molecule which does not display the appropriate epitope(s). Preferably the affinity of the antibody will be at least about 5 fold, preferably 10 fold, more preferably 25-fold, even more preferably 50-fold, and most preferably 100-fold or more, greater for a target molecule than its affinity for a non-target molecule. In preferred embodiments, Preferred antibodies bind with affinities of at least about 107 M"1, and preferably between about 108 M"1 to about 109 M"1, about 109 M"1 to about 1010 M"1, or about 1010 M"1 to about 1012 M"1 . [0096] Affinity is calculated as Kd = koff/kon (koff is the dissociation rate constant, K0n is the association rate constant and Kd is the equilibrium constant). Affinity can be determined at equilibrium by measuring the fraction bound (r) of labeled ligand at various concentrations (c). The data are graphed using the Scatchard equation: r/c = K(n-r): where r = moles of bound ligand/mole of receptor at equilibrium; c = free ligand concentration at equilibrium; K = equilibrium association constant; and n = number of ligand binding sites per receptor molecule. By graphical analysis, r/c is plotted on the Y-axis versus r on the X- axis, thus producing a Scatchard plot. Antibody affinity measurement by Scatchard analysis is well known in the art. See, e.g., van Erp et al, J. Immunoassay 12: 425-43, 1991; Nelson and Griswold, Comput. Methods Programs Biomed. 27: 65-8, 1988.
[0097] The term "epitope" refers to an antigenic determinant capable of specific binding to an antibody. Epitopes usually consist of chemically active surface groupings of molecules such as amino acids or sugar side chains and usually have specific three dimensional structural characteristics, as well as specific charge characteristics. Conformational and nonconformational epitopes are distinguished in that the binding to the former but not the latter is lost in the presence of denaturing solvents.
[0098] Numerous publications discuss the use of phage display technology to produce and screen libraries of polypeptides for binding to a selected analyte. See, e.g, Cwirla et al., Proc. Natl. Acad. ScL USA 87, 6378-82, 1990; Devlin et al, Science 249, 404-6, 1990, Scott and Smith, Science 249, 386-88, 1990; and Ladner et al, U.S. Pat. No. 5,571,698. A basic concept of phage display methods is the establishment of a physical association between DNA encoding a polypeptide to be screened and the polypeptide. This physical association is provided by the phage particle, which displays a polypeptide as part of a capsid enclosing the phage genome which encodes the polypeptide. The establishment of a physical association between polypeptides and their genetic material allows simultaneous mass screening of very large numbers of phage bearing different polypeptides. Phage displaying a polypeptide with affinity to a target bind to the target and these phage are enriched by affinity screening to the target. The identity of polypeptides displayed from these phage can be determined from their respective genomes. Using these methods a polypeptide identified as having a binding affinity for a desired target can then be synthesized in bulk by conventional means. See, e.g., U.S. Patent No. 6,057,098, which is hereby incorporated in its entirety, including all tables, figures, and claims. [0099] The antibodies that are generated by these methods may then be selected by first screening for affinity and specificity with the purified polypeptide of interest and, if required, comparing the results to the affinity and specificity of the antibodies with polypeptides that are desired to be excluded from binding. The screening procedure can involve immobilization of the purified polypeptides in separate wells of microtiter plates. The solution containing a potential antibody or groups of antibodies is then placed into the respective microtiter wells and incubated for about 30 min to 2 h. The microtiter wells are then washed and a labeled secondary antibody (for example, an anti-mouse antibody conjugated to alkaline phosphatase if the raised antibodies are mouse antibodies) is added to the wells and incubated for about 30 min and then washed. Substrate is added to the wells and a color reaction will appear where antibody to the immobilized polypeptide(s) are present.
[0100] The antibodies so identified may then be further analyzed for affinity and specificity in the assay design selected. In the development of immunoassays for a target protein, the purified target protein acts as a standard with which to judge the sensitivity and specificity of the immunoassay using the antibodies that have been selected. Because the binding affinity of various antibodies may differ; certain antibody pairs (e.g., in sandwich assays) may interfere with one another sterically, etc., assay performance of an antibody may be a more important measure than absolute affinity and specificity of an antibody.
Assay Correlations
[0101] The term "correlating" as used herein in reference to the use of biomarkers refers to comparing the presence or amount of the biomarker(s) in a patient to its presence or amount in persons known to suffer from, or known to be at risk of, a given condition; or in persons known to be free of a given condition. Often, this takes the form of comparing an assay result in the form of a biomarker concentration to a predetermined threshold selected to be indicative of the occurrence or nonoccurrence of a disease or the likelihood of some future outcome.
[0102] Selecting a diagnostic threshold involves, among other things, consideration of the probability of disease, distribution of true and false diagnoses at different test thresholds, and estimates of the consequences of treatment (or a failure to treat) based on the diagnosis. For example, when considering administering a specific therapy which is highly efficacious and has a low level of risk, few tests are needed because clinicians can accept substantial diagnostic uncertainty. On the other hand, in situations where treatment options are less effective and more risky, clinicians often need a higher degree of diagnostic certainty. Thus, cost/benefit analysis is involved in selecting a diagnostic threshold.
[0103] Suitable thresholds may be determined in a variety of ways. For example, one recommended diagnostic threshold for the diagnosis of acute myocardial infarction using cardiac troponin is the 97.5th percentile of the concentration seen in a normal population. Another method may be to look at serial samples from the same patient, where a prior "baseline" result is used to monitor for temporal changes in a biomarker level.
[0104] Population studies may also be used to select a decision threshold. Reciever Operating Characteristic ("ROC") arose from the field of signal dectection therory developed during World War II for the analysis of radar images, and ROC analysis is often used to select a threshold able to best distinguish a "diseased" subpopulation from a "nondiseased" subpopulation. A false positive in this case occurs when the person tests positive, but actually does not have the disease. A false negative, on the other hand, occurs when the person tests negative, suggesting they are healthy, when they actually do have the disease. To draw a ROC curve, the true positive rate (TPR) and false positive rate (FPR) are determined as the decision threshold is varied continuously. Since TPR is equivalent with sensitivity and FPR is equal to 1 - specificity, the ROC graph is sometimes called the sensitivity vs (1 - specificity) plot. A perfect test will have an area under the ROC curve of 1.0; a random test will have an area of 0.5. A threshold is selected to provide an acceptable level of specificity and sensitivity.
[0105] In this context, "diseased" is meant to refer to a population having one characteristic (the presence of a disease or condition or the occurrence of some outcome) and "nondiseased" is meant to refer to a population lacking the characteristic. While a single decision threshold is the simplest application of such a method, multiple decision thresholds may be used. For example, below a first threshold, the absence of disease may be assigned with relatively high confidence, and above a second threshold the presence of disease may also be assigned with relatively high confidence. Between the two thresholds may be considered indeterminate. This is meant to be exemplary in nature only.
[0106] In addition to threshold comparisons, other methods for correlating assay results to a patient classification (occurrence or nonoccurrence of disease, likelihood of an outcome, etc.) include decision trees, rule sets, Bayesian methods, and neural network methods. These methods can produce probability values representing the degree to which a subject belongs to one classification out of a plurality of classifications.
[0107] Measures of test accuracy may be obtained as described in Fischer et al, Intensive Care Med. 29: 1043-51, 2003, and used to determine the effectiveness of a given biomarker. These measures include sensitivity and specificity, predictive values, likelihood ratios, diagnostic odds ratios, and ROC curve areas. The area under the curve ("AUC") of a ROC plot is equal to the probability that a classifier will rank a randomly chosen positive instance higher than a randomly chosen negative one. The area under the ROC curve may be thought of as equivalent to the Mann-Whitney U test, which tests for the median difference between scores obtained in the two groups considered if the groups are of continuous data, or to the Wilcoxon test of ranks.
[0108] As discussed above, suitable tests may exhibit one or more of the following results on these various measures: a specificity of greater than 0.5, preferably at least 0.6, more preferably at least 0.7, still more preferably at least 0.8, even more preferably at least 0.9 and most preferably at least 0.95, with a corresponding sensitivity greater than 0.2, preferably greater than 0.3, more preferably greater than 0.4, still more preferably at least 0.5, even more preferably 0.6, yet more preferably greater than 0.7, still more preferably greater than 0.8, more preferably greater than 0.9, and most preferably greater than 0.95; a sensitivity of greater than 0.5, preferably at least 0.6, more preferably at least 0.7, still more preferably at least 0.8, even more preferably at least 0.9 and most preferably at least 0.95, with a corresponding specificity greater than 0.2, preferably greater than 0.3, more preferably greater than 0.4, still more preferably at least 0.5, even more preferably 0.6, yet more preferably greater than 0.7, still more preferably greater than 0.8, more preferably greater than 0.9, and most preferably greater than 0.95; at least 75% sensitivity, combined with at least 75% specificity; a ROC curve area of greater than 0.5, preferably at least 0.6, more preferably 0.7, still more preferably at least 0.8, even more preferably at least 0.9, and most preferably at least 0.95; an odds ratio different from 1, preferably at least about 2 or more or about 0.5 or less, more preferably at least about 3 or more or about 0.33 or less, still more preferably at least about 4 or more or about 0.25 or less, even more preferably at least about 5 or more or about 0.2 or less, and most preferably at least about 10 or more or about 0.1 or less; a positive likelihood ratio (calculated as sensitivity/(l -specificity)) of greater than 1, at least 2, more preferably at least 3, still more preferably at least 5, and most preferably at least 10; and or a negative likelihood ratio (calculated as (1 -sensitivity )/specificity) of less than 1, less than or equal to 0.5, more preferably less than or equal to 0.3, and most preferably less than or equal to 0.1
[0109] Additional clinical indicia may be combined with the kidney injury marker assay result(s) of the present invention. These include other biomarkers related to renal status. Examples include the following, which recite the common biomarker name, followed by the Swiss-Prot entry number for that biomarker or its parent: Actin (P68133); Adenosine deaminase binding protein (DPP4, P27487); Alpha- 1-acid glycoprotein 1 (P02763); Alpha- 1 -microglobulin (P02760); Albumin (P02768); Angiotensinogenase (Renin, P00797); Annexin A2 (P07355); Beta-glucuronidase (P08236); B-2-microglobulin (P61679); Beta- galactosidase (P16278); BMP-7 (P18075); Brain natriuretic peptide (proBNP, BNP-32, NTproBNP; P16860); Calcium-binding protein Beta (SlOO-beta, P04271); Carbonic anhydrase (Q16790); Casein Kinase 2 (P68400); Cathepsin B (P07858); Ceruloplasmin (P00450); Clusterin (P10909); Complement C3 (P01024); Cysteine-rich protein (CYR61, 000622); Cytochrome C (P99999); Epidermal growth factor (EGF, POl 133); Endothelin-1 (P05305); Exosomal Fetuin-A (P02765); Fatty acid-binding protein, heart (FABP3, P05413); Fatty acid-binding protein, liver (P07148); Ferritin (light chain, P02793; heavy chain P02794); Fructose- 1,6-biphosphatase (P09467); GRO-alpha (CXCLl, (P09341); Growth Hormone (P01241); Hepatocyte growth factor (P14210); Insulin-like growth factor I (PO 1343); Immunoglobulin G; Immunoglobulin Light Chains (Kappa and Lambda); Interferon gamma (P01308); Lysozyme (P61626); Interleukin-1 alpha (P01583); Interleukin- 2 (P60568); Interleukin-4 (P60568); Interleukin-9 (P15248); Interleukin-12p40 (P29460); Interleukin-13 (P35225); Interleukin-16 (Q14005); Ll cell adhesion molecule (P32004); Lactate dehydrogenase (P00338); Leucine Aminopeptidase (P28838); Meprin A-alpha subunit (Q16819); Meprin A-beta subunit (Q16820); Midkine (P21741); MIP2-alpha (CXCL2, P19875); MMP-2 (P08253); MMP-9 (P14780); Netrin-1 (095631); Neutral endopeptidase (P08473); Osteopontin (P10451); Renal papillary antigen 1 (RPAl); Renal papillary antigen 2 (RP A2); Retinol binding protein (P09455); Ribonuclease; SlOO calcium- binding protein A6 (P06703); Serum Amyloid P Component (P02743); Sodium/Hydrogen exchanger isoform (NHE3, P48764); Spermidine/spermine Nl-acetyltransferase (P21673); TGF-Betal (POl 137); Transferrin (P02787); Trefoil factor 3 (TFF3, Q07654); Toll-Like protein 4 (000206); Total protein; Tubulointerstitial nephritis antigen (Q9UJW2); Uromodulin (Tamm-Horsfall protein, P07911). [0110] For purposes of risk stratification, Adiponectin (Q15848); Alkaline phosphatase (P05186); Aminopeptidase N (P15144); CalbindinD28k (P05937); Cystatin C (P01034); 8 subunit of FIFO ATPase (P03928); Gamma-glutamyltransferase (P19440); GSTa (alpha- glutathione-S-transferase, P08263); GSTpi (Glutathione-S-transferase P; GST class-pi; P09211); IGFBP-I (P08833); IGFBP-2 (P18065); IGFBP-6 (P24592); Integral membrane protein 1 (Itml, P46977); Interleukin-6 (P05231); Interleukin-8 (P10145); Interleukin-18 (Q14116); IP-IO (10 kDa interferon-gamma-induced protein, P02778); IRPR (IFRDl, 000458); Isovaleryl-CoA dehydrogenase (IVD, P26440); I-TAC/CXCL11 (014625); Keratin 19 (P08727); Kim-1 (Hepatitis A virus cellular receptor 1, 043656); L- arginine: glycine amidinotransferase (P50440); Leptin (P41159); Lipocalin2 (NGAL, P80188); MCP-I (P13500); MIG (Gamma-interferon-induced monokine Q07325); MIP-Ia (P10147); MIP-3a (P78556); MIP-lbeta (P13236); MIP-Id (Q16663); NAG (N-acetyl-beta- D-glucosaminidase, P54802); Organic ion transporter (OCT2, 015244); Osteoprotegerin (014788); P8 protein (060356); Plasminogen activator inhibitor 1 (PAI-I, P05121); ProANP(l-98) (POl 160); Protein phosphatase 1-beta (PPI-beta, P62140); Rab GDI-beta (P50395); Renal kallikrein (Q86U61 ); RTl.B-1 (alpha) chain of the integral membrane protein (Q5Y7A8); Soluble tumor necrosis factor receptor superfamily member IA (sTNFR- I, P19438); Soluble tumor necrosis factor receptor superfamily member IB (sTNFR-II, P20333); Tissue inhibitor of metalloproteinases 3 (TEVIP-3, P35625); uPAR (Q03405) may be combined with the kidney injury marker assay result(s) of the present invention.
[0111] Other clinical indicia which may be combined with the kidney injury marker assay result(s) of the present invention includes demographic information (e.g., weight, sex, age, race), medical history (e.g., family history, type of surgery, pre-existing disease such as aneurism, congestive heart failure, preeclampsia, eclampsia, diabetes mellitus, hypertension, coronary artery disease, proteinuria, renal insufficiency, or sepsis, type of toxin exposure such as NSAIDs, cyclosporines, tacrolimus, aminoglycosides, foscarnet, ethylene glycol, hemoglobin, myoglobin, ifosfamide, heavy metals, methotrexate, radiopaque contrast agents, or strep tozotocin), clinical variables (e.g., blood pressure, temperature, respiration rate), risk scores (APACHE score, PREDICT score, TIMI Risk Score for UA/NSTEMI, Framingham Risk Score), a urine total protein measurement, a glomerular filtration rate, an estimated glomerular filtration rate, a urine production rate, a serum or plasma creatinine concentration, a renal papillary antigen 1 (RPAl) measurement; a renal papillary antigen 2 (RP A2) measurement; a urine creatinine concentration, a fractional excretion of sodium, a urine sodium concentration, a urine creatinine to serum or plasma creatinine ratio, a urine specific gravity, a urine osmolality, a urine urea nitrogen to plasma urea nitrogen ratio, a plasma BUN to creatnine ratio, and/or a renal failure index calculated as urine sodium / (urine creatinine / plasma creatinine). Other measures of renal function which may be combined with the kidney injury marker assay result(s) are described hereinafter and in Harrison's Principles of Internal Medicine, 17th Ed., McGraw Hill, New York, pages 1741- 1830, and Current Medical Diagnosis & Treatment 2008, 47th Ed, McGraw Hill, New York, pages 785-815, each of which are hereby incorporated by reference in their entirety.
[0112] Combining assay results/clinical indicia in this manner can comprise the use of multivariate logistical regression, loglinear modeling, neural network analysis, n-of-m analysis, decision tree analysis, etc. This list is not meant to be limiting.
[0113] Diagnosis of Acute Renal Failure
[0114] As noted above, the terms "acute renal (or kidney) injury" and "acute renal (or kidney) failure" as used herein are defined in part in terms of changes in serum creatinine from a baseline value. Most definitions of ARF have common elements, including the use of serum creatinine and, often, urine output. Patients may present with renal dysfunction without an available baseline measure of renal function for use in this comparison. In such an event, one may estimate a baseline serum creatinine value by assuming the patient initially had a normal GFR. Glomerular filtration rate (GFR) is the volume of fluid filtered from the renal (kidney) glomerular capillaries into the Bowman's capsule per unit time. Glomerular filtration rate (GFR) can be calculated by measuring any chemical that has a steady level in the blood, and is freely filtered but neither reabsorbed nor secreted by the kidneys. GFR is typically expressed in units of ml/min:
Urine Concentration x Urine Flow
GFi?
Plasma Concentration
[0115] By normalizing the GFR to the body surface area, a GFR of approximately 75- 100 ml/min per 1.73 m2 can be assumed. The rate therefore measured is the quantity of the substance in the urine that originated from a calculable volume of blood.
[0116] There are several different techniques used to calculate or estimate the glomerular filtration rate (GFR or eGFR). In clinical practice, however, creatinine clearance is used to measure GFR. Creatinine is produced naturally by the body (creatinine is a metabolite of creatine, which is found in muscle). It is freely filtered by the glomerulus, but also actively secreted by the renal tubules in very small amounts such that creatinine clearance overestimates actual GFR by 10-20%. This margin of error is acceptable considering the ease with which creatinine clearance is measured.
[0117] Creatinine clearance (CCr) can be calculated if values for creatinine's urine concentration (UQ-), urine flow rate (V), and creatinine's plasma concentration (PCr) are known. Since the product of urine concentration and urine flow rate yields creatinine's excretion rate, creatinine clearance is also said to be its excretion rate (UcrxV) divided by its plasma concentration. This is commonly represented mathematically as:
Figure imgf000051_0001
[0118] Commonly a 24 hour urine collection is undertaken, from empty-bladder one morning to the contents of the bladder the following morning, with a comparative blood test then taken:
Figure imgf000051_0002
[0119] To allow comparison of results between people of different sizes, the CCr is often corrected for the body surface area (BSA) and expressed compared to the average sized man as ml/min/1.73 m2. While most adults have a BSA that approaches 1.7 (1.6-1.9), extremely obese or slim patients should have their CCr corrected for their actual BSA:
(. -Q1. X l . t O
C - Or —r'oιτe:c.tt:% BSA
[0120] The accuracy of a creatinine clearance measurement (even when collection is complete) is limited because as glomerular filtration rate (GFR) falls creatinine secretion is increased, and thus the rise in serum creatinine is less. Thus, creatinine excretion is much greater than the filtered load, resulting in a potentially large overestimation of the GFR (as much as a twofold difference). However, for clinical purposes it is important to determine whether renal function is stable or getting worse or better. This is often determined by monitoring serum creatinine alone. Like creatinine clearance, the serum creatinine will not be an accurate reflection of GFR in the non-steady-state condition of ARF. Nonetheless, the degree to which serum creatinine changes from baseline will reflect the change in GFR. Serum creatinine is readily and easily measured and it is specific for renal function.
[0121] For purposes of determining urine output on a Urine output on a mL/kg/hr basis, hourly urine collection and measurement is adequate. In the case where, for example, only a cumulative 24-h output was available and no patient weights are provided, minor modifications of the RIFLE urine output criteria have been described. For example, Bagshaw et ah, Nephrol. Dial. Transplant. 23: 1203-1210, 2008, assumes an average patient weight of 70 kg, and patients are assigned a RIFLE classification based on the following: <35 mL/h (Risk), <21 mL/h (Injury) or <4 mL/h (Failure).
[0122] Selecting a Treatment Regimen
[0123] Once a diagnosis is obtained, the clinician can readily select a treatment regimen that is compatible with the diagnosis, such as initiating renal replacement therapy, withdrawing delivery of compounds that are known to be damaging to the kidney, kidney transplantation, delaying or avoiding procedures that are known to be damaging to the kidney, modifying diuretic administration, initiating goal directed therapy, etc. The skilled artisan is aware of appropriate treatments for numerous diseases discussed in relation to the methods of diagnosis described herein. See, e.g., Merck Manual of Diagnosis and Therapy, 17th Ed. Merck Research Laboratories, Whitehouse Station, NJ, 1999. In addition, since the methods and compositions described herein provide prognostic information, the markers of the present invention may be used to monitor a course of treatment. For example, improved or worsened prognostic state may indicate that a particular treatment is or is not efficacious.
[0124] One skilled in the art readily appreciates that the present invention is well adapted to carry out the objects and obtain the ends and advantages mentioned, as well as those inherent therein. The examples provided herein are representative of preferred embodiments, are exemplary, and are not intended as limitations on the scope of the invention.
[0125] Example 1: Contrast-induced nephropathy sample collection
[0126] The objective of this sample collection study is to collect samples of plasma and urine and clinical data from patients before and after receiving intravascular contrast media. Approximately 250 adults undergoing radiographic/angiographic procedures involving intravascular administration of iodinated contrast media are enrolled. To be enrolled in the study, each patient must meet all of the following inclusion criteria and none of the following exclusion criteria: Inclusion Criteria males and females 18 years of age or older; undergoing a radiographic / angiographic procedure (such as a CT scan or coronary intervention) involving the intravascular administration of contrast media; expected to be hospitalized for at least 48 hours after contrast administration. able and willing to provide written informed consent for study participation and to comply with all study procedures.
Exclusion Criteria renal transplant recipients; acutely worsening renal function prior to the contrast procedure; already receiving dialysis (either acute or chronic) or in imminent need of dialysis at enrollment; expected to undergo a major surgical procedure (such as involving cardiopulmonary bypass) or an additional imaging procedure with contrast media with significant risk for further renal insult within the 48 hrs following contrast administration; participation in an interventional clinical study with an experimental therapy within the previous 30 days; known infection with human immunodeficiency virus (HIV) or a hepatitis virus.
[0127] Immediately prior to the first contrast administration (and after any pre-procedure hydration), an EDTA anti-coagulated blood sample (10 mL) and a urine sample (10 mL) are collected from each patient. Blood and urine samples are then collected at 4 (±0.5), 8 (±1), 24 (±2) 48 (±2), and 72 (±2) hrs following the last administration of contrast media during the index contrast procedure. Blood is collected via direct venipuncture or via other available venous access, such as an existing femoral sheath, central venous line, peripheral intravenous line or hep-lock. These study blood samples are processed to plasma at the clinical site, frozen and shipped to Astute Medical, Inc., San Diego, CA. The study urine samples are frozen and shipped to Astute Medical, Inc.
[0128] Serum creatinine is assessed at the site immediately prior to the first contrast administration (after any pre-procedure hydration) and at 4 (±0.5), 8 (±1), 24 (±2) and 48 (±2) ), and 72 (±2) hours following the last administration of contrast (ideally at the same time as the study samples are obtained). In addition, each patient's status is evaluated through day 30 with regard to additional serum and urine creatinine measurements, a need for dialysis, hospitalization status, and adverse clinical outcomes (including mortality).
[0129] Prior to contrast administration, each patient is assigned a risk based on the following assessment: systolic blood pressure <80 mm Hg = 5 points; intra- arterial balloon pump = 5 points; congestive heart failure (Class III- IV or history of pulmonary edema) = 5 points; age >75 yrs = 4 points; hematocrit level <39% for men, <35% for women = 3 points; diabetes = 3 points; contrast media volume = 1 point for each 100 mL; serum creatinine level >1.5 g/dL = 4 points OR estimated GFR 40-60 niL/min/1.73 m2 = 2 points, 20-40 niL/min/1.73 m2 = 4 points, < 20 niL/min/1.73 m2 = 6 points. The risks assigned are as follows: risk for CIN and dialysis: 5 or less total points = risk of CIN - 7.5%, risk of dialysis - 0.04%; 6-10 total points = risk of CIN - 14%, risk of dialysis - 0.12%; 11-16 total points = risk of CIN - 26.1%, risk of dialysis - 1.09%; >16 total points = risk of CIN - 57.3%, risk of dialysis - 12.8%.
[0130] Example 2: Cardiac surgery sample collection
[0131] The objective of this sample collection study is to collect samples of plasma and urine and clinical data from patients before and after undergoing cardiovascular surgery, a procedure known to be potentially damaging to kidney function. Approximately 900 adults undergoing such surgery are enrolled. To be enrolled in the study, each patient must meet all of the following inclusion criteria and none of the following exclusion criteria:
Inclusion Criteria males and females 18 years of age or older; undergoing cardiovascular surgery;
Toronto/Ottawa Predictive Risk Index for Renal Replacement risk score of at least 2 (Wijeysundera et al, JAMA 297: 1801-9, 2007); and able and willing to provide written informed consent for study participation and to comply with all study procedures.
Exclusion Criteria known pregnancy; previous renal transplantation; acutely worsening renal function prior to enrollment (e.g., any category of RIFLE criteria); already receiving dialysis (either acute or chronic) or in imminent need of dialysis at enrollment; currently enrolled in another clinical study or expected to be enrolled in another clinical study within 7 days of cardiac surgery that involves drug infusion or a therapeutic intervention for AKI; known infection with human immunodeficiency virus (HIV) or a hepatitis virus.
[0132] Within 3 hours prior to the first incision (and after any pre -procedure hydration), an EDTA anti-coagulated blood sample (10 mL), whole blood (3 mL), and a urine sample (35 mL) are collected from each patient. Blood and urine samples are then collected at 3 (±0.5), 6 (±0.5), 12 (±1), 24 (±2) and 48 (±2) hrs following the procedure and then daily on days 3 through 7 if the subject remains in the hospital. Blood is collected via direct venipuncture or via other available venous access, such as an existing femoral sheath, central venous line, peripheral intravenous line or hep-lock. These study blood samples are frozen and shipped to Astute Medical, Inc., San Diego, CA. The study urine samples are frozen and shipped to Astute Medical, Inc.
[0133] Example 3: Acutely ill subject sample collection
[0134] The objective of this study is to collect samples from acutely ill patients. Approximately 900 adults expected to be in the ICU for at least 48 hours will be enrolled. To be enrolled in the study, each patient must meet all of the following inclusion criteria and none of the following exclusion criteria:
Inclusion Criteria males and females 18 years of age or older;
Study population 1: approximately 300 patients that have at least one of: shock (SBP < 90 mmHg and/or need for vasopressor support to maintain MAP > 60 mmHg and/or documented drop in SBP of at least 40 mmHg); and sepsis;
Study population 2: approximately 300 patients that have at least one of: IV antibiotics ordered in computerized physician order entry (CPOE) within 24 hours of enrollment; contrast media exposure within 24 hours of enrollment; increased Intra- Abdominal Pressure with acute decompensated heart failure; and severe trauma as the primary reason for ICU admission and likely to be hospitalized in the ICU for 48 hours after enrollment;
Study population 3: approximately 300 patients expected to be hospitalized through acute care setting (ICU or ED) with a known risk factor for acute renal injury (e.g. sepsis, hypotension/shock (Shock = systolic BP < 90 mmHg and/or the need for vasopressor support to maintain a MAP > 60 mmHg and/or a documented drop in SBP > 40 mmHg), major trauma, hemorrhage, or major surgery); and/or expected to be hospitalized to the ICU for at least 24 hours after enrollment.
Exclusion Criteria known pregnancy; institutionalized individuals; previous renal transplantation; known acutely worsening renal function prior to enrollment (e.g., any category of RIFLE criteria); received dialysis (either acute or chronic) within 5 days prior to enrollment or in imminent need of dialysis at the time of enrollment; known infection with human immunodeficiency virus (HIV) or a hepatitis virus; meets only the SBP < 90 mmHg inclusion criterion set forth above, and does not have shock in the attending physician's or principal investigator's opinion.
[0135] After providing informed consent, an EDTA anti-coagulated blood sample (10 mL) and a urine sample (25-30 mL) are collected from each patient. Blood and urine samples are then collected at 4 (± 0.5) and 8 (± 1) hours after contrast administration (if applicable); at 12 (± 1), 24 (± 2), and 48 (± 2) hours after enrollment, and thereafter daily up to day 7 to day 14 while the subject is hospitalized. Blood is collected via direct venipuncture or via other available venous access, such as an existing femoral sheath, central venous line, peripheral intravenous line or hep-lock. These study blood samples are processed to plasma at the clinical site, frozen and shipped to Astute Medical, Inc., San Diego, CA. The study urine samples are frozen and shipped to Astute Medical, Inc.
[0136] Example 4. Immunoassay format
[0137] Analytes are is measured using standard sandwich enzyme immunoassay techniques. A first antibody which binds the analyte is immobilized in wells of a 96 well polystyrene microplate. Analyte standards and test samples are pipetted into the appropriate wells and any analyte present is bound by the immobilized antibody. After washing away any unbound substances, a horseradish peroxidase-conjugated second antibody which binds the analyte is added to the wells, thereby forming sandwich complexes with the analyte (if present) and the first antibody. Following a wash to remove any unbound antibody-enzyme reagent, a substrate solution comprising tetramethylbenzidine and hydrogen peroxide is added to the wells. Color develops in proportion to the amount of analyte present in the sample. The color development is stopped and the intensity of the color is measured at 540 nm or 570 nm. An analyte concentration is assigned to the test sample by comparison to a standard curve determined from the analyte standards.
[0138] Concentrations are expressed in the following examples as follows: soluble p- selectin - ng/mL; protein NOV homolog - pg.mL; netrin 4 - ng/mL; haptoglobin - mg/mL; alpha- 1 -antitrypsin - mg/mL; leukocyte elastase - ng/mL; soluble tumor necrosis factor receptor superfamily member 6 - pg/mL; soluble tumor necrosis factor ligand superfamily member 6 - pg/mL; soluble intercellular adhesion molecule 2 - units/mL; caspase 3 (active) - ng/mL; soluble platelet endothelial cell adhesion molecule - ng/mL; heat shock protein beta-1 - ng/mL; soluble epidermal growth factor receptor - pg/mL.
[0139] Example 5. Apparently Healthy Donor and Chronic Disease Patient
Samples
[0140] Human urine samples from donors with no known chronic or acute disease ("Apparently Healthy Donors") were purchased from two vendors (Golden West Biologicals, Inc., 27625 Commerce Center Dr., Temecula, CA 92590 and Virginia Medical Research, Inc., 915 First Colonial Rd., Virginia Beach, VA 23454). The urine samples were shipped and stored frozen at less than -20° C. The vendors supplied demographic information for the individual donors including gender, race (Black /White), smoking status and age. [0141] Human urine samples from donors with various chronic diseases ("Chronic Disease Patients") including congestive heart failure, coronary artery disease, chronic kidney disease, chronic obstructive pulmonary disease, diabetes mellitus and hypertension were purchased from Virginia Medical Research, Inc., 915 First Colonial Rd., Virginia Beach, VA 23454. The urine samples were shipped and stored frozen at less than -20 degrees centigrade. The vendor provided a case report form for each individual donor with age, gender, race (Black/White), smoking status and alcohol use, height, weight, chronic disease(s) diagnosis, current medications and previous surgeries.
[0142] Example 6. Kidney injury markers for evaluating renal status in patients at
RIFLE Stage 0
[0143] Patients from the intensive care unit (ICU) were classified by kidney status as non-injury (0), risk of injury (R), injury (I), and failure (F) according to the maximum stage reached within 7 days of enrollment as determined by the RIFLE criteria.
[0144] Two cohorts were defined as (Cohort 1) patients that did not progress beyond stage 0, and (Cohort 2) patients that reached stage R, I, or F within 10 days. To address normal marker fluctuations that occur within patients at the ICU and thereby assess utility for monitoring AKI status, marker levels in urine samples collected for Cohort 1. Marker concentrations were measured in urine samples collected from a subject at 0, 24 hours, and 48 hours prior to reaching stage R, I or F in Cohort 2. In the following tables, the time "prior max stage" represents the time at which a sample is collected, relative to the time a particular patient reaches the lowest disease stage as defined for that cohort, binned into three groups which are +/- 12 hours. For example, 24 hr prior for this example (0 vs R, I, F) would mean 24 hr (+/- 12 hours) prior to reaching stage R (or I if no sample at R, or F if no sample at R or I).
[0145] Each marker was measured by standard immunoassay methods using commercially available assay reagents. A receiver operating characteristic (ROC) curve was generated for each marker and the area under each ROC curve (AUC) was determined. Patients in Cohort 2 were also separated according to the reason for adjudication to stage R, I, or F as being based on serum creatinine measurements (sCr), being based on urine output (UO), or being based on either serum creatinine measurements or urine output. That is, for those patients adjudicated to stage R, I, or F on the basis of serum creatinine measurements alone, the stage 0 cohort may have included patients adjudicated to stage R, I, or F on the basis of urine output; for those patients adjudicated to stage R, I, or F on the basis of urine output alone, the stage 0 cohort may have included patients adjudicated to stage R, I, or F on the basis of serum creatinine measurements; and for those patients adjudicated to stage R, I, or F on the basis of serum creatinine measurements or urine output, the stage 0 cohort contains only patients in stage 0 for both serum creatinine measurements and urine output. Also, for those patients adjudicated to stage R, I, or F on the basis of serum creatinine measurements or urine output, the adjudication method which yielded the most severe RIFLE stage was used.
[0146] The following descriptive statistics were obtained: Soluble p-selectin: sCr or UO
0 hr prior to AKI stage 24 hr prior to AKI stage 48 hr prior to AKI stage
Cohort Cohort
Cohort 1 2 Cohort 1 2 Cohort 1 Cohort 2 median 0 .244 0.183 0.244 0.290 0.244 0 000 average 0 .243 0.217 0.243 3.764 0.243 0 190 stdev 0 .153 0.162 0.153 16.703 0.153 na p (t-test) 0.527 0.134 na min 0 .000 0.000 0.000 0.000 0.000 0 190 max 0 .662 0.662 0.662 82.167 0.662 0 190 n (Samp) 51 22 51 24 51 1 n (Pat) 40 22 40 24 40 1
sCr only
Figure imgf000059_0001
Figure imgf000060_0001
Protein NOV homolog: sCr or UO
0 hr prior to AKI stage 24 hr prior to AKI stage 48 hr prior to AKI stage
Cohort 1 Cohort 2 Cohort 1 Cohort 2 Cohort 1 Cohort 2 median 25253.378 52184.874 25253.378 60843.958 25253.378 66053.687 average 43022.422 62430.121 43022.422 83958.436 43022.422 66053.687 stdev 46997.024 56276.741 46997.024 70276.919 46997.024 23004.666 p (t-test) 0.057 0.001 0.492 min 14.544 1226.994 14.544 3448.276 14.544 49786.932 max 227486.911 211725.664 227486.911 228010.471 227486.911 82320.442 n (Samp) 101 31 101 26 101 2 n (Pat) 50 31 50 26 50 2
sCr only
Figure imgf000060_0002
Figure imgf000061_0001
Netrin 4:
Figure imgf000061_0002
sCr only
Figure imgf000061_0003
Figure imgf000062_0001
Haptoglobin:
Figure imgf000062_0002
sCr only
0 hr prior to AKI stage 24 hr prior to AKI stage 48 hr prior to AKI stage
Cohort 1 Cohort 2 Cohort 1 Cohort 2 Cohort 1 Cohort 2 median 0.000 0.000 0.000 0.000 0.000 0.000 average 0.001 0.000 0.001 0.000 0.001 0.000 stdev 0.001 0.000 0.001 0.001 0.001 0.000 p (t-test) 0.403 0.705 0.560 min 0.000 0.000 0.000 0.000 0.000 0.000 max 0.007 0.002 0.007 0.003 0.007 0.001 n (Samp) 375 16 375 21 375 11 n (Pat) 127 16 127 21 127 11
Figure imgf000063_0001
Alpha- 1 -antitrypsin:
Figure imgf000063_0002
sCr only
0 hr prior toAKI stage 24 hr prior to AKI stage 48 hr prior to AKI stage
Cohort 1 ( Cohort 2 Cohort 1 Cohort 2 Cohort 1 Cohort 2 median 0 .001 0.001 0.001 0.001 0.001 0.001 average 0 .001 0.001 0.001 0.001 0.001 0.001 stdev 0 .001 0.001 0.001 0.001 0.001 0.001 p (t-test) 0.093 0.063 0.261 min 0 .000 0.000 0.000 0.000 0.000 0.000 max 0 .005 0.002 0.005 0.003 0.005 0.003 n (Samp) 375 16 375 21 375 11 n (Pat) 127 16 127 21 127 11
Figure imgf000064_0001
Leukocyte elastase: sCr or
Figure imgf000064_0002
sCr only
0 hr prior to AKI stage 24 hr prior to AKI stage 48 hr ] Drior to AKI stage
Cohort 1 Cohort 2 Cohort 1 Cohort 2 Cohort 1 Cohort 2 median 34 568 33 123 34.568 31. 179 34 568 43 691 average 43 827 42 392 43 827 38. 160 43 827 55 480 stdev 37 819 36 409 37 819 38. 649 37 819 44 934 p (t-test) 0 898 0. 542 0 324 min 0 080 3 965 0 080 2. 174 0 080 1 002 max 137 517 103 409 137 517 125. 675 137 517 125 810 n (Samp) 226 12 226 18 226 11 n (Pat) 106 12 106 18 106 11
Figure imgf000065_0001
Soluble tumor necrosis factor receptor superfamilv member 6:
Figure imgf000065_0002
sCr only
Figure imgf000065_0003
Figure imgf000066_0001
Soluble tumor necrosis factor ligand superfamilv member 6:
Figure imgf000066_0002
sCr only
Figure imgf000066_0003
Figure imgf000067_0001
Soluble intercellular adhesion molecule 2:
Figure imgf000067_0002
sCr only
Figure imgf000067_0003
Figure imgf000068_0001
Caspase 3 (active):
Figure imgf000068_0002
sCr only
Figure imgf000068_0003
Figure imgf000069_0001
Soluble platelet endothelial cell adhesion molecule:
Figure imgf000069_0002
sCr only
Figure imgf000069_0003
Figure imgf000070_0001
Heat shock protein beta- 1 :
Figure imgf000070_0002
sCr only
Figure imgf000070_0003
Figure imgf000071_0001
Soluble epidermal growth factor receptor:
Figure imgf000071_0002
sCr only
Figure imgf000071_0003
Figure imgf000072_0001
[0147] In the following tables, the ability to distinguish cohort 1 (subjects remaining in RIFLE 0) from Cohort 2 (subjects progressing to RIFLE R, I or F) was determined using ROC analysis. SE is the standard error of the AUC, n is the number of sample or individual patients ("pts," as indicated). Standard errors were calculated as described in Hanley, J. A., and McNeil, B. J., The meaning and use of the area under a receiver operating characteristic (ROC) curve. Radiology (1982) 143: 29-36; p values were calculated with a two-tailed Z- test. An AUC < 0.5 is indicative of a negative going marker for the comparison, and an AUC > 0.5 is indicative of a positive going marker for the comparison.
Soluble p-selectin:
Cohort 1 v Cohort 2, adjudicated on serum creatinine measurements or urine output
Figure imgf000072_0002
Cohort 1 v Cohort 2, adjudicated on serum creatinine
Figure imgf000072_0003
Cohort 1 v Cohort 2, adjudicated on urine output
Figure imgf000073_0001
Protein NOV homolog:
Cohort 1 v Cohort 2, adjudicated on serum creatinine measurements or urine output
Figure imgf000073_0002
Cohort 1 v Cohort 2, adjudicated on serum creatinine
Figure imgf000073_0003
Cohort 1 v Cohort 2, adjudicated on urine output
Figure imgf000073_0004
Netrin 4:
Cohort 1 v Cohort 2, adjudicated on serum creatinine measurements or urine output
Figure imgf000074_0001
Cohort 1 v Cohort 2, adjudicated on serum creatinine
Figure imgf000074_0002
Cohort 1 v Cohort 2, adjudicated on urine output
Figure imgf000074_0003
Haptoglobin:
Cohort 1 v Cohort 2, adjudicated on serum creatinine measurements or urine output
Figure imgf000074_0004
Cohort 1 v Cohort 2, adjudicated on serum creatinine
Figure imgf000075_0001
Cohort 1 v Cohort 2, adjudicated on urine output
Figure imgf000075_0002
Alpha- 1 -antitrypsin:
Cohort 1 v Cohort 2, adjudicated on serum creatinine measurements or urine output
Figure imgf000075_0003
Cohort 1 v Cohort 2, adjudicated on serum creatinine
Figure imgf000075_0004
Cohort 1 v Cohort 2, adjudicated on urine output
Figure imgf000075_0005
Leukocyte elastase:
Cohort 1 v Cohort 2, adjudicated on serum creatinine measurements or urine output
Figure imgf000076_0001
Cohort 1 v Cohort 2, adjudicated on serum creatinine
Figure imgf000076_0002
Cohort 1 v Cohort 2, adjudicated on urine output
Figure imgf000076_0003
Soluble tumor necrosis factor receptor superfamily member 6:
Cohort 1 v Cohort 2, adjudicated on serum creatinine measurements or urine output
Figure imgf000076_0004
Cohort 1 v Cohort 2, adjudicated on serum creatinine
Figure imgf000077_0001
Cohort 1 v Cohort 2, adjudicated on urine output
Figure imgf000077_0002
Soluble tumor necrosis factor ligand superfamily member 6:
Cohort 1 v Cohort 2, adjudicated on serum creatinine measurements or urine output
Figure imgf000077_0003
Cohort 1 v Cohort 2, adjudicated on serum creatinine
Figure imgf000077_0004
Cohort 1 v Cohort 2, adjudicated on urine output
Figure imgf000077_0005
Soluble intercellular adhesion molecule 2:
Cohort 1 v Cohort 2, adjudicated on serum creatinine measurements or urine output
Figure imgf000078_0001
Cohort 1 v Cohort 2, adjudicated on serum creatinine
Figure imgf000078_0002
Cohort 1 v Cohort 2, adjudicated on urine output
Figure imgf000078_0003
Caspase 3 (active):
Cohort 1 v Cohort 2, adjudicated on serum creatinine measurements or urine output
Figure imgf000078_0004
Cohort 1 v Cohort 2, adjudicated on serum creatinine
Figure imgf000079_0001
Cohort 1 v Cohort 2, adjudicated on urine output
Figure imgf000079_0002
Soluble platelet endothelial cell adhesion molecule:
Cohort 1 v Cohort 2, adjudicated on serum creatinine measurements or urine output
Figure imgf000079_0003
Cohort 1 v Cohort 2, adjudicated on serum creatinine
Figure imgf000079_0004
Cohort 1 v Cohort 2, adjudicated on urine output
Figure imgf000079_0005
Heat shock protein beta- 1 :
Cohort 1 v Cohort 2, adjudicated on serum creatinine measurements or urine output
Figure imgf000080_0001
Cohort 1 v Cohort 2, adjudicated on serum creatinine
Figure imgf000080_0002
Cohort 1 v Cohort 2, adjudicated on urine output
Figure imgf000080_0003
Soluble epidermal growth factor receptor:
Cohort 1 v Cohort 2, adjudicated on serum creatinine measurements or urine output
Figure imgf000080_0004
Cohort 1 v Cohort 2, adjudicated on serum creatinine
Figure imgf000081_0001
Cohort 1 v Cohort 2, adjudicated on urine output
[0148] Various threshold (or "cutoff) concentrations were selected, and the associated sensitivity and specificity for distinguishing cohort 1 from cohort 2, as shown in the following tables. OR is the odds ratio calculated for the particular cutoff concentration, and 95% CI is the confidence interval for the odds ratio.
Soluble p-selectin: sCr or UO
Figure imgf000081_0003
0.44303694 0% 90% sCr only
Figure imgf000082_0002
Figure imgf000082_0001
Figure imgf000082_0003
Figure imgf000083_0001
Protein NOV homolog: sCr or UO
Figure imgf000083_0002
sCr only
Figure imgf000083_0003
Figure imgf000084_0001
Figure imgf000084_0002
Netrin 4: sCr or UO
Figure imgf000085_0001
sCr only
Figure imgf000085_0002
Figure imgf000086_0001
Haptoglobin: sCr or UO
Figure imgf000086_0002
Figure imgf000087_0001
Figure imgf000087_0002
Figure imgf000087_0003
Alpha- 1 -antitrypsin: sCr or UO
Figure imgf000088_0001
sCr onl
Figure imgf000088_0002
Figure imgf000089_0001
Leukocyte elastase: sCr or UO
Figure imgf000089_0002
Cr only
Figure imgf000090_0001
Figure imgf000090_0002
Soluble tumor necrosis factor receptor superfamily member 6: sCr or UO
Figure imgf000091_0001
sCr onl
Figure imgf000091_0002
Figure imgf000092_0001
Soluble tumor necrosis factor ligand superfamilv member 6: sCr or UO
Figure imgf000092_0002
Figure imgf000093_0002
sCr only
Figure imgf000093_0003
Figure imgf000093_0001
Figure imgf000093_0004
Figure imgf000094_0001
Soluble intercellular adhesion molecule 2: sCr or UO
Figure imgf000094_0002
sCr only
Figure imgf000094_0003
Figure imgf000095_0001
Figure imgf000095_0002
Caspase 3 (active): sCr or UO
Figure imgf000096_0001
sCr onl
Figure imgf000096_0002
Figure imgf000097_0001
Soluble platelet endothelial cell adhesion molecule: sCr or UO
Figure imgf000097_0002
Figure imgf000098_0002
sCr only
Figure imgf000098_0003
Figure imgf000098_0001
Figure imgf000098_0004
Figure imgf000099_0001
Heat shock protein beta- 1 : sCr or UO
Figure imgf000099_0002
sCr onl
Figure imgf000099_0003
Figure imgf000100_0001
Figure imgf000100_0002
Soluble epidermal growth factor receptor: sCr or UO
Figure imgf000100_0003
Figure imgf000101_0002
sCr only
Figure imgf000101_0003
Figure imgf000101_0001
Figure imgf000101_0004
Figure imgf000102_0002
[0149] Example 7. Kidney injury markers for evaluating renal status in patients at
RIFLE Stages 0 and R
[0150] Patients were classified and analyzed as described in Example 6. However, patients that reached stage R but did not progress to stage I or F were grouped with patients from non-injury stage 0 in Cohort 1. Cohort 2 in this example included only patients that progressed to stage I or F. Marker concentrations in urine samples were included for Cohort 1. Marker concentrations in urine samples collected within 0, 24, and 48 hours of reaching stage I or F were included for Cohort 2.
[0151] The following descriptive statistics were obtained: Soluble p-selectin:
Figure imgf000102_0001
sCr only
Figure imgf000103_0001
Figure imgf000103_0002
Protein NOV homolog:
Figure imgf000103_0003
sCr only
Figure imgf000104_0001
UO only
0 hr prior to AKI stage 24 hr prior to AKI stage 48 hr prior to AKI stage
Cohort 1 Cohort 2 Cohort 1 Cohort 2 Cohort 1 Cohort 2 median 38281.250 37922.297 38281.250 38168.932 38281.250 29478.827 average 48607.167 43800.740 48607.167 67966.748 48607.167 50816.935 stdev 45556.799 42686.735 45556.799 69694.118 45556.799 60224.503 p (t-test) 0.759 0.134 0.934 min 14.544 1785.714 14.544 9600.515 14.544 4166.667 max 226963.351 139120.879 226963.351 228010.471 226963.351 118805.310 n (Samp) 133 9 133 16 133 3 n (Pat) 71 9 71 16 71 3
Netrin 4:
Figure imgf000104_0002
sCr only
Figure imgf000105_0001
Alpha- 1 -antitrypsin: sCr or UO
0 hr prior to AKI stage 24 hr prior to AKI stage 48 hr prior to AKI stage
Cohort 1 Cohort 2 Cohort 1 Cohort 2 Cohort 1 Cohort 2 median 0.001 0.001 0.001 0.001 0.001 0.000 average 0.001 0.001 0.001 0.001 0.001 0.001 stdev 0.001 0.001 0.001 0.001 0.001 0.001 p (t-test) 0.467 0.240 0.286 min 0.000 0.000 0.000 0.000 0.000 0.000 max 0.005 0.003 0.005 0.003 0.005 0.003 n (Samp) 351 21 351 25 351 13 n (Pat) 121 21 121 25 121 13 sCr only
0 hr prior to AKI stage 24 hr prior to AKI stage 48 hr prior to AKI stage
Cohort 1 Cohort 2 Cohort 1 Cohort 2 Cohort 1 Cohort 2 median 0.001 0.001 0.001 0.001 0.001 0.001 average 0.001 0.001 0.001 0.001 0.001 0.001 stdev 0.001 0.001 0.001 0.001 0.001 0.001 p (t-test) 0.509 0.301 0.224 min 0.000 0.000 0.000 0.000 0.000 0.000 max 0.005 0.002 0.005 0.003 0.005 0.002 n (Samp) 428 5 428 6 428 5 n (Pat) 146 5 | 146 146 5 |
Figure imgf000106_0001
Leukocyte elastase: sCr or UO
0 hr prior to AKI stage 24 hr prior to AKI stage 48 hr prior to AKI stage
Cohort 1 Cohort 2 Cohort 1 Cohort 2 Cohort 1 Cohort 2 median 27.160 60.358 27.160 78.767 27.160 61.235 average 37.230 57.770 37.230 69.139 37.230 48.020 stdev 34.627 46.896 34.627 40.418 34.627 37.264 p (t-test) 0.024 0.000 0.279 min 0.080 0.678 0.080 2.106 0.080 0.899 max 137.517 136.336 137.517 125.592 137.517 110.014 n (Samp) 202 17 202 23 202 13 n (Pat) 106 17 106 23 106 13 sCr only
Figure imgf000106_0002
Figure imgf000107_0001
Soluble tumor necrosis factor receptor superfamilv member 6:
Figure imgf000107_0002
sCr only
Figure imgf000107_0003
Figure imgf000108_0001
Soluble tumor necrosis factor ligand superfamilv member 6:
Figure imgf000108_0002
sCr only
Figure imgf000108_0003
Figure imgf000109_0001
Soluble intercellular adhesion molecule 2:
Figure imgf000109_0002
sCr only
Figure imgf000109_0003
Figure imgf000110_0001
Heat shock protein beta- 1 :
Figure imgf000110_0002
sCr only
Figure imgf000110_0003
Figure imgf000111_0001
In the following tables, the ability to distinguish cohort 1 (subjects remaining in RIFLE 0 or R) from Cohort 2 (subjects progressing to RIFLE I or F) was determined using ROC analysis.
Soluble p-selectin:
Cohort 1 v Cohort 2, adjudicated on serum creatinine measurements or urine output
Figure imgf000111_0002
Cohort 1 v Cohort 2, adjudicated on serum creatinine
Figure imgf000111_0003
Cohort 1 v Cohort 2, adjudicated on urine output
Figure imgf000111_0004
Protein NOV homolog:
Cohort 1 v Cohort 2, adjudicated on serum creatinine measurements or urine output
Figure imgf000112_0001
Cohort 1 v Cohort 2, adjudicated on serum creatinine
Figure imgf000112_0002
Cohort 1 v Cohort 2, adjudicated on urine output
Figure imgf000112_0003
Netrin 4:
Cohort 1 v Cohort 2, adjudicated on serum creatinine measurements or urine output
Figure imgf000112_0004
Cohort 1 v Cohort 2, adjudicated on serum creatinine
Figure imgf000113_0001
Cohort 1 v Cohort 2, adjudicated on urine output
Figure imgf000113_0002
Alpha- 1 -antitrypsin:
Cohort 1 v Cohort 2, adjudicated on serum creatinine measurements or urine output
Figure imgf000113_0003
Cohort 1 v Cohort 2, adjudicated on serum creatinine
Figure imgf000113_0004
Cohort 1 v Cohort 2, adjudicated on urine output
Figure imgf000113_0005
Leukocyte elastase:
Cohort 1 v Cohort 2, adjudicated on serum creatinine measurements or urine output
Figure imgf000114_0001
Cohort 1 v Cohort 2, adjudicated on serum creatinine
Figure imgf000114_0002
Cohort 1 v Cohort 2, adjudicated on urine output
Figure imgf000114_0003
Soluble tumor necrosis factor receptor superfamily member 6:
Cohort 1 v Cohort 2, adjudicated on serum creatinine measurements or urine output
Figure imgf000114_0004
Cohort 1 v Cohort 2, adjudicated on serum creatinine
Figure imgf000114_0005
Figure imgf000115_0001
Cohort 1 v Cohort 2, adjudicated on urine output
Figure imgf000115_0002
Soluble tumor necrosis factor ligand superfamily member 6:
Cohort 1 v Cohort 2, adjudicated on serum creatinine measurements or urine output
Figure imgf000115_0003
Cohort 1 v Cohort 2, adjudicated on serum creatinine
Figure imgf000115_0004
Cohort 1 v Cohort 2, adjudicated on urine output
Figure imgf000115_0005
Soluble intercellular adhesion molecule 2:
Cohort 1 v Cohort 2, adjudicated on serum creatinine measurements or urine output
Figure imgf000116_0001
Cohort 1 v Cohort 2, adjudicated on serum creatinine
Figure imgf000116_0002
Cohort 1 v Cohort 2, adjudicated on urine output
Figure imgf000116_0003
Heat shock protein beta- 1 :
Cohort 1 v Cohort 2, adjudicated on serum creatinine measurements or urine output
Figure imgf000116_0004
Cohort 1 v Cohort 2, adjudicated on serum creatinine
Figure imgf000116_0005
Cohort 1 v Cohort 2, adjudicated on urine output
Figure imgf000117_0001
[0152] Various threshold (or "cutoff) concentrations were selected, and the associated sensitivity and specificity for distinguishing cohort 1 from cohort 2, as shown in the following tables. OR is the odds ratio calculated for the particular cutoff concentration, and 95% CI is the confidence interval for the odds ratio.
Soluble p-selectin:
sCr or UO
Figure imgf000117_0002
sCr only
Figure imgf000117_0003
Figure imgf000118_0001
Figure imgf000118_0002
Protein NOV homolog:
sCr or UO
Figure imgf000119_0001
sCr onl
Figure imgf000119_0002
Figure imgf000120_0001
Netrin 4: sCr or UO
Figure imgf000120_0002
Figure imgf000121_0002
sCr only
Figure imgf000121_0003
Figure imgf000121_0001
Figure imgf000121_0004
Alpha- 1 -antitrypsin:
sCr or UO
Figure imgf000122_0001
sCr only
Figure imgf000122_0002
Figure imgf000123_0001
Leukocyte elastase: sCr or UO
Figure imgf000123_0002
90.38461538 15% 90% sCr only
Figure imgf000124_0002
Figure imgf000124_0001
Figure imgf000124_0003
Soluble tumor necrosis factor receptor superfamily member 6: sCr or UO
Figure imgf000125_0001
sCr only
Figure imgf000125_0002
Figure imgf000126_0001
Soluble tumor necrosis factor ligand superfamilv member 6: sCr or UO
Figure imgf000126_0002
Cr only
Figure imgf000127_0001
Figure imgf000127_0002
Soluble intercellular adhesion molecule 2: sCr or UO
Figure imgf000128_0001
sCr onl
Figure imgf000128_0002
Figure imgf000129_0001
Heat shock protein beta- 1 : sCr or UO
Figure imgf000129_0002
Figure imgf000130_0001
sCr only
Figure imgf000130_0002
Figure imgf000131_0001
[0153] Example 8. Kidney injury markers for evaluating renal status in patients progressing from Stage R to Stages I and F
[0154] Patients were classified and analyzed as described in Example 6, but only those patients that reached Stage R were included in this example. Cohort 1 contained patients that reached stage R but did not progress to stage I or F within 10 days, and Cohort 2 included only patients that progressed to stage I or F. Marker concentrations in urine samples collected within 12 hours of reaching stage R were included in the analysis for both Cohort 1 and 2. [0155] The following descriptive statistics were obtained Alpha- 1 -antitrypsin:
Figure imgf000132_0001
sCr only
Figure imgf000132_0002
UO only
0 hr prior to AKI stage 24 hr prior to AKI stage 48 hr prior to AKI stage
Cohort 1 Cohort 2 Cohort 1 Cohort 2 Cohort 1 Cohort 2 median 0.001 0.001 0.001 0.001 0.001 0.001 average 0.001 0.001 0.001 0.001 0.001 0.001 stdev 0.001 0.001 0.001 0.001 0.001 0.001 p (t-test) 0.179 0.179 0.179 min 0.000 0.000 0.000 0.000 0.000 0.000 max 0.005 0.002 0.005 0.002 0.005 0.002 n (Samp) 25 13 25 13 25 13 n (Pat) 25 13 25 13 25 13 Leukocyte elastase:
Figure imgf000133_0001
sCr only
Figure imgf000133_0002
Soluble tumor necrosis factor receptor superfamily member 6:
Figure imgf000134_0001
Cr only
Figure imgf000134_0002
UO only
0 hr prior to AKI stage 24 hr prior to AKI stage 48 hr prior to AKI stage
Cohort 1 Cohort 2 Cohort 1 Cohort 2 Cohort 1 Cohort 2 median 615.549 416.168 615.549 416.168 615.549 416.168 average 663.825 582.789 663.825 582.789 663.825 582.789 stdev 445.268 477.453 445.268 477.453 445.268 477.453 p (t-test) 0.671 0.671 0.671 min 108.491 225.000 108.491 225.000 108.491 225.000 max 1846.785 1648.865 1846.785 1648.865 1846.785 1648.865 n (Samp) 21 8 21 8 21 8 n (Pat) 21 8 21 8 21 8
[0156] In the following tables, the ability to distinguish cohort 1 (subjects remaining in RIFLE R) from Cohort 2 (subjects progressing to RIFLE I or F) was determined using ROC analysis. Alpha- 1 -antitrypsin:
Cohort 1 v Cohort 2, adjudicated on serum creatinine measurements or urine output
Figure imgf000135_0001
Cohort 1 v Cohort 2, adjudicated on serum creatinine
Figure imgf000135_0002
Cohort 1 v Cohort 2, adjudicated on urine output
Figure imgf000135_0003
Leukocyte elastase:
Cohort 1 v Cohort 2, adjudicated on serum creatinine measurements or urine output
Figure imgf000135_0004
Cohort 1 v Cohort 2, adjudicated on serum creatinine
Figure imgf000136_0001
Cohort 1 v Cohort 2, adjudicated on urine output
Figure imgf000136_0002
Soluble tumor necrosis factor receptor superfamily member 6:
Cohort 1 v Cohort 2, adjudicated on serum creatinine measurements or urine output
Figure imgf000136_0003
Cohort 1 v Cohort 2, adjudicated on serum creatinine
Figure imgf000136_0004
Cohort 1 v Cohort 2, adjudicated on urine output
Figure imgf000136_0005
[0157] Various threshold (or "cutoff) concentrations were selected, and the associated sensitivity and specificity for distinguishing cohort 1 from cohort 2, as shown in the following tables. OR is the odds ratio calculated for the particular cutoff concentration, and 95% CI is the confidence interval for the odds ratio:
Alpha- 1 -antitrypsin: sCr or UO
Figure imgf000137_0001
sCr only
Figure imgf000137_0002
Figure imgf000138_0001
Figure imgf000138_0002
Leukocyte elastase: sCr or UO
Figure imgf000139_0001
sCr only
Figure imgf000139_0002
Figure imgf000140_0001
Soluble tumor necrosis factor receptor superfamilv member 6: sCr or UO
Figure imgf000140_0002
Figure imgf000141_0002
sCr only
Figure imgf000141_0003
Figure imgf000141_0001
Figure imgf000141_0004
[0158] Example 9. Kidney injury markers for evaluating renal status in patients at
RIFLE Stage 0
[0159] Patients from the intensive care unit (ICU) were classified by kidney status as non-injury (0), risk of injury (R), injury (I), and failure (F) according to the maximum stage reached within 7 days of enrollment as determined by the RIFLE criteria.
[0160] Two cohorts were defined as (Cohort 1) patients that did not progress beyond stage 0, and (Cohort 2) patients that reached stage R, I, or F within 10 days. To address normal marker fluctuations that occur within patients at the ICU and thereby assess utility for monitoring AKI status, marker levels in blood samples collected for Cohort 1. Marker concentrations were measured in the plasma component of the blood samples collected from a subject at 0, 24 hours, and 48 hours prior to reaching stage R, I or F in Cohort 2. In the following tables, the time "prior max stage" represents the time at which a sample is collected, relative to the time a particular patient reaches the lowest disease stage as defined for that cohort, binned into three groups which are +/- 12 hours. For example, 24 hr prior for this example (0 vs R, I, F) would mean 24 hr (+/- 12 hours) prior to reaching stage R (or I if no sample at R, or F if no sample at R or I).
[0161] Each marker was measured by standard immunoassay methods using commercially available assay reagents. A receiver operating characteristic (ROC) curve was generated for each marker and the area under each ROC curve (AUC) was determined. Patients in Cohort 2 were also separated according to the reason for adjudication to stage R, I, or F as being based on serum creatinine measurements (sCr), being based on urine output (UO), or being based on either serum creatinine measurements or urine output. That is, for those patients adjudicated to stage R, I, or F on the basis of serum creatinine measurements alone, the stage 0 cohort may have included patients adjudicated to stage R, I, or F on the basis of urine output; for those patients adjudicated to stage R, I, or F on the basis of urine output alone, the stage 0 cohort may have included patients adjudicated to stage R, I, or F on the basis of serum creatinine measurements; and for those patients adjudicated to stage R, I, or F on the basis of serum creatinine measurements or urine output, the stage 0 cohort contains only patients in stage 0 for both serum creatinine measurements and urine output. Also, for those patients adjudicated to stage R, I, or F on the basis of serum creatinine measurements or urine output, the adjudication method which yielded the most severe RIFLE stage was used. [0162] The following descriptive statistics were obtained: Soluble p-selectin:
Figure imgf000143_0001
sCr only
Figure imgf000143_0002
UO only
0 hr prior to AKI stage 24 hr prior to AKI stage 48 hr prior to AKI stage
Cohort 1 Cohort 2 Cohort 1 Cohort 2 Cohort 1 Cohort 2 median 71.039 96.643 71.039 69.671 71.039 26.445 average 69.479 87.362 69.479 76.898 69.479 71.409 stdev 32.279 26.376 32.279 43.557 32.279 na p (t-test) 0.187 0.566 na min 26.445 56.486 26.445 27.323 26.445 71.409 max 148.265 119.078 148.265 195.149 148.265 71.409 n (Samp) 27 7 27 11 27 1 n (Pat) 25 7 25 11 25 1 Protein NOV homolog:
sCr or UO
0 hr prior to AKI stage 24 hr prior to AKI stage 48 hr prior to AKI sta^ Ψ
Cohort 1 Cohort 2 Cohort 1 Cohort 2 Cohort 1 Cohort 2 median 7867.816 10136.555 7867.816 11134.454 7867.816 14. 544 average 32628.823 11619.711 32628.823 11746.019 32628.823 13928. 571 stdev 118993.020 7490.315 118993.020 9009.294 118993.020 na p (t-test) 0.422 0.436 na min 14.544 1362.398 14.544 730.519 14.544 13928. 571 max 1005084.746 29632.588 1005084.746 32110.092 1005084.746 13928. 571 n (Samp) 82 21 82 20 82 1 n (Pat) 47 21 47 20 47 1 sCr only
0 hr prior to AKI stage 24 hr prior to AKI stage 48 hr prior to AKI stage
Cohort 1 Cohort 2 Cohort 1 Cohort 2 Cohort 1 Cohort 2 median 8560.924 15515.485 8560.924 11263.342 8560.924 21232.057 average 24336.406 20501.633 24336.406 13302.341 24336.406 28699.397 stdev 93316.115 16311.708 93316.115 9797.524 93316.115 19613.823 p (t-test) 0.908 0.684 0.936 min 14.544 1362.398 14.544 730.519 14.544 13916.016 max 1005084.746 54988.124 1005084.746 39449.541 1005084.746 50950.119 n (Samp) 135 8 135 12 135 3 n (Pat) 78 8 78 12 78 3
UO only
0 hr prior to AKI stage 24 hr prior to AKI stage 48 hr prior to AKI stage
Cohort 1 Cohort 2 Cohort 1 Cohort 2 Cohort 1 Cohort 2 median 9195.402 12646.484 9195.402 11449.580 9195.402 13629.325 average 36969.265 13836.073 36969.265 15641.491 36969.265 13629.325 stdev 130178.851 8858.174 130178.851 13967.881 130178.851 423.199 p (t-test) 0.420 0.544 0.802 min 14.544 1542.208 14.544 974.026 14.544 13330.078 max 1005084.746 32827.476 1005084.746 48955.614 1005084.746 13928.571 n (Samp) 68 21 68 14 68 2 n (Pat) 37 21 37 14 37 2
Netrin 4:
Figure imgf000145_0001
sCr only
Figure imgf000145_0002
Haptoglobin:
Figure imgf000146_0001
sCr only
0 hr prior to AKI stage 24 hr prior to AKI stage 48 hr prior to AKI stage
Cohort 1 Cohort 2 Cohort 1 Cohort 2 Cohort 1 Cohort 2 median 2.465 1.640 2.465 1.910 2.465 1.985 average 2.759 2.213 2.759 2.473 2.759 1.804 stdev 2.083 2.136 2.083 2.067 2.083 0.796 p (t-test) 0.306 0.540 0.149 min 0.000 0.000 0.000 0.000 0.000 0.703 max 12.700 8.320 12.700 8.870 12.700 2.730 n (Samp) 386 16 386 21 386 10 n (Pat) 129 16 129 21 129 10
Figure imgf000146_0002
Alpha- 1 -antitrypsin:
Figure imgf000147_0001
sCr only
0 hr prior to AKI stage 24 hr prior to AKI stage 48 hr prior to AKI stage
Cohort 1 Cohort 2 Cohort 1 Cohort 2 Cohort 1 Cohort 2 median 3.040 2.505 3.040 2.390 3.040 2.670 average 3.311 2.854 3.311 2.558 3.311 2.701 stdev 1.455 1.465 1.455 1.354 1.455 1.386 p (t-test) 0.220 0.021 0.191 min 0.769 1.220 0.769 0.968 0.769 0.737 max 8.930 6.700 8.930 5.580 8.930 5.060 n (Samp) 386 16 386 21 386 10 n (Pat) 129 16 129 21 129 10
Figure imgf000147_0002
Leukocyte elastase:
Figure imgf000148_0001
sCr only
Figure imgf000148_0002
Soluble tumor necrosis factor receptor superfamily member 6:
Figure imgf000149_0001
sCr only
Figure imgf000149_0002
UO only
0 hr prior to AKI stage 24 hr prior to AKI stage 48 hr prior to AKI stage Cohort 1 Cohort 2 Cohort 1 Cohort 2 Cohort 1 Cohort 2 median 8909.217 12729.659 8909.217 10828.803 8909.217 9831.547 average 9531.137 12459.768 9531.137 11236.077 9531.137 10169.720 stdev 3917.481 4106.211 3917.481 3615.748 3917.481 3481.582 p (t-test) 0.010 0.112 0.782 min 3586.165 6493.109 3586.165 6171.516 3586.165 6869.565 max 20557.276 18935.447 20557.276 18369.196 20557.276 13808.050 n (Samp) 78 15 78 16 78 3 n (Pat) 29 15 29 16 29 3
Soluble tumor necrosis factor ligand superfamily member 6:
Figure imgf000150_0001
sCr only
Figure imgf000150_0002
Soluble intercellular adhesion molecule 2:
Figure imgf000151_0001
sCr only
Figure imgf000151_0002
UO only
0 hr prior to AKI stage 24 hr prior to AKI stage 48 hr prior to AKI stage
Cohort 1 Cohort 2 Cohort 1 Cohort 2 Cohort 1 Cohort 2 median 354.483 392.230 354.483 272.118 354.483 na average 492.930 482.921 492.930 312.819 492.930 na stdev 322.471 281.363 322.471 130.973 322.471 na p (t-test) 0.925 0.349 na min 34.681 115.768 34.681 207.029 34.681 na max 1343.415 920.780 1343.415 459.310 1343.415 na n (Samp) 33 12 33 3 33 0 n (Pat) 16 12 16 3 16 0
Soluble platelet endothelial cell adhesion molecule:
Figure imgf000152_0001
sCr only
Figure imgf000152_0002
UO only
0 hr prior to AKI stage 24 hr prior to AKI stage 48 hr prior to AKI stage
Cohort 1 Cohort 2 Cohort 1 Cohort 2 Cohort 1 Cohort 2 median 73.464 78.271 73.464 52.986 73.464 na average 75.845 85.411 75.845 62.838 75.845 na stdev 26.038 45.034 26.038 22.404 26.038 na p (t-test) 0.380 0.410 na min 42.620 39.576 42.620 47.048 42.620 na max 158.173 193.269 158.173 88.480 158.173 na n (Samp) 33 12 33 3 33 0 n (Pat) 16 12 16 3 16 0
Heat shock protein beta- 1 :
Figure imgf000153_0001
sCr only
Figure imgf000153_0002
UO only
0 hr prior to AKI stage 24 hr prior to AKI stage 48 hr prior to AKI stage
Cohort 1 Cohort 2 Cohort 1 Cohort 2 Cohort 1 Cohort 2 median 88.186 35.469 88.186 57.533 88.186 14.768 average 136.091 57.330 136.091 178.691 136.091 16.349 stdev 130.907 57.559 130.907 243.575 130.907 na p (t-test) 0.029 0.609 na min 14.768 8.406 14.768 19.454 14.768 16.349 max 683.847 211.848 683.847 459.086 683.847 16.349 n (Samp) 42 15 42 3 42 1 n (Pat) 27 15 27 3 27 1
[0163] In the following tables, the ability to distinguish cohort 1 (subjects remaining in RIFLE 0) from Cohort 2 (subjects progressing to RIFLE R, I or F) was determined using ROC analysis. SE is the standard error of the AUC, n is the number of sample or individual patients ("pts," as indicated). Standard errors were calculated as described in Hanley, J. A., and McNeil, B. J., The meaning and use of the area under a receiver operating characteristic (ROC) curve. Radiology (1982) 143: 29-36; p values were calculated with a two-tailed Z- test. An AUC < 0.5 is indicative of a negative going marker for the comparison, and an AUC > 0.5 is indicative of a positive going marker for the comparison.
Soluble p-selectin:
Cohort 1 v Cohort 2, adjudicated on serum creatinine measurements or urine output
Figure imgf000154_0001
Cohort 1 v Cohort 2, adjudicated on serum creatinine
Figure imgf000154_0002
Cohort 1 v Cohort 2, adjudicated on urine output
Figure imgf000154_0003
Protein NOV homolog:
Cohort 1 v Cohort 2, adjudicated on serum creatinine measurements or urine output
Figure imgf000155_0001
Cohort 1 v Cohort 2, adjudicated on serum creatinine
Figure imgf000155_0002
Cohort 1 v Cohort 2, adjudicated on urine output
Figure imgf000155_0003
Netrin 4:
Cohort 1 v Cohort 2, adjudicated on serum creatinine measurements or urine output
Figure imgf000155_0004
Cohort 1 v Cohort 2, adjudicated on serum creatinine
Figure imgf000156_0001
Cohort 1 v Cohort 2, adjudicated on urine output
Figure imgf000156_0002
Haptoglobin:
Cohort 1 v Cohort 2, adjudicated on serum creatinine measurements or urine output
Figure imgf000156_0003
Cohort 1 v Cohort 2, adjudicated on serum creatinine
Figure imgf000156_0004
Cohort 1 v Cohort 2, adjudicated on urine output
Figure imgf000156_0005
Alpha- 1 -antitrypsin:
Cohort 1 v Cohort 2, adjudicated on serum creatinine measurements or urine output
Figure imgf000157_0001
Cohort 1 v Cohort 2, adjudicated on serum creatinine
Figure imgf000157_0002
Cohort 1 v Cohort 2, adjudicated on urine output
Figure imgf000157_0003
Leukocyte elastase:
Cohort 1 v Cohort 2, adjudicated on serum creatinine measurements or urine output
Figure imgf000157_0004
Cohort 1 v Cohort 2, adjudicated on serum creatinine
Figure imgf000158_0001
Cohort 1 v Cohort 2, adjudicated on urine output
Figure imgf000158_0002
Soluble tumor necrosis factor receptor superfamily member 6:
Cohort 1 v Cohort 2, adjudicated on serum creatinine measurements or urine output
Figure imgf000158_0003
Cohort 1 v Cohort 2, adjudicated on serum creatinine
Figure imgf000158_0004
Cohort 1 v Cohort 2, adjudicated on urine output
Figure imgf000158_0005
Soluble tumor necrosis factor ligand superfamily member 6:
Cohort 1 v Cohort 2, adjudicated on serum creatinine measurements or urine output
Figure imgf000159_0001
Cohort 1 v Cohort 2, adjudicated on serum creatinine
Figure imgf000159_0002
Cohort 1 v Cohort 2, adjudicated on urine output
Figure imgf000159_0003
Soluble intercellular adhesion molecule 2:
Cohort 1 v Cohort 2, adjudicated on serum creatinine measurements or urine output
Figure imgf000159_0004
Cohort 1 v Cohort 2, adjudicated on serum creatinine
Figure imgf000160_0001
Cohort 1 v Cohort 2, adjudicated on urine output
Figure imgf000160_0002
Soluble platelet endothelial cell adhesion molecule:
Cohort 1 v Cohort 2, adjudicated on serum creatinine measurements or urine output
Figure imgf000160_0003
Cohort 1 v Cohort 2, adjudicated on serum creatinine
Figure imgf000160_0004
Cohort 1 v Cohort 2, adjudicated on urine output
Figure imgf000160_0005
Heat shock protein beta- 1 :
Cohort 1 v Cohort 2, adjudicated on serum creatinine measurements or urine output
Figure imgf000161_0001
Cohort 1 v Cohort 2, adjudicated on serum creatinine
Figure imgf000161_0002
Cohort 1 v Cohort 2, adjudicated on urine output
Figure imgf000161_0003
[0164] Various threshold (or "cutoff) concentrations were selected, and the associated sensitivity and specificity for distinguishing cohort 1 from cohort 2, as shown in the following tables. OR is the odds ratio calculated for the particular cutoff concentration, and 95% CI is the confidence interval for the odds ratio. Soluble p-selectin: sCr or UO
Figure imgf000162_0001
sCr only
Figure imgf000162_0002
Figure imgf000163_0001
Protein NOV homolog: sCr or UO
Figure imgf000163_0002
Figure imgf000164_0002
sCr only
Figure imgf000164_0003
Figure imgf000164_0001
Figure imgf000164_0004
Netrin 4: sCr or UO
Figure imgf000165_0001
sCr only
Figure imgf000165_0002
Figure imgf000166_0001
Haptoglobin: sCr or UO
Figure imgf000166_0002
Cr onl
Figure imgf000167_0002
Figure imgf000167_0001
Figure imgf000167_0003
Figure imgf000168_0001
Alpha- 1 -antitrypsin: sCr or UO
Figure imgf000168_0002
sCr onl
Figure imgf000168_0003
Figure imgf000169_0001
Leukocyte elastase: sCr or UO
Figure imgf000169_0002
Figure imgf000170_0002
sCr only
Figure imgf000170_0003
Figure imgf000170_0001
Figure imgf000170_0004
Soluble tumor necrosis factor receptor superfamily member 6: sCr or UO
Figure imgf000171_0001
sCr only
Figure imgf000171_0002
Figure imgf000172_0001
Soluble tumor necrosis factor ligand superfamilv member 6: sCr or UO
Figure imgf000172_0002
Cr only
Figure imgf000173_0002
Figure imgf000173_0001
Figure imgf000173_0003
Soluble intercellular adhesion molecule 2: sCr or UO
Figure imgf000174_0001
sCr only
Figure imgf000174_0002
Figure imgf000175_0001
Soluble platelet endothelial cell adhesion molecule: sCr or UO
Figure imgf000175_0002
Cr onl
Figure imgf000176_0002
Figure imgf000176_0001
Figure imgf000176_0003
Heat shock protein beta- 1 : sCr or UO
Figure imgf000177_0001
sCr onl
Figure imgf000177_0002
Figure imgf000178_0001
[0165] Example 10. Kidney injury markers for evaluating renal status in patients at
RIFLE Stages 0 and R
[0166] Patients were classified and analyzed as described in Example 9. However, patients that reached stage R but did not progress to stage I or F were grouped with patients from non-injury stage 0 in Cohort 1. Cohort 2 in this example included only patients that progressed to stage I or F. Marker concentrations in the plasma component of blood samples were included for Cohort 1. Marker concentrations the plasma component of blood samples collected within 0, 24, and 48 hours of reaching stage I or F were included for Cohort 2.
[0167] The following descriptive statistics were obtained: Soluble p-selectin:
Figure imgf000179_0001
sCr only
Figure imgf000179_0002
UO only
0 hr prior to AKI stage 24 hr prior to AKI stage 48 hr prior to AKI stage
Cohort 1 Cohort 2 Cohort 1 Cohort 2 Cohort 1 Cohort 2 median 70.355 na 70.355 59.964 70.355 26.445 average 75.995 na 75.995 65.611 75.995 71.409 stdev 40.724 na 40.724 27.299 40.724 na p (t-test) na 0.396 na min 26.445 na 26.445 24.586 26.445 71.409 max 196.760 na 196.760 115.156 196.760 71.409 n (Samp) 40 0 40 13 40 1 n (Pat) 37 0 37 13 37 1
Protein NOV homolog: sCr or UO
0 hr prior to AKI stage 24 hr prior to AKI stage 48 hr prior to AKI stage
Cohort 1 Cohort 2 Cohort 1 Cohort 2 Cohort 1 Cohort 2 median 9608.193 10919.540 9608.193 10031.513 9608.193 13217.703 average 25945.538 17907.223 25945.538 10960.289 25945.538 11866.502 stdev 96388.752 20623.132 96388.752 8150.749 96388.752 2438.316 p (t-test) 0.804 0.524 0.801 min 14.544 1668.937 14.544 974.026 14.544 9051.724 max 1005084.746 66983.373 1005084.746 28035.144 1005084.746 13330.078 n (Samp) 126 9 126 17 126 3 n (Pat) 73 9 73 17 73 3 sCr only
Figure imgf000180_0001
Netrin 4:
Figure imgf000180_0002
sCr only
0 hr prior to AKI stage 24 hr prior to AKI stage 48 hr prior to AKI stage
Cohort 1 Cohort 2 Cohort 1 Cohort 2 Cohort 1 Cohort 2 median 0.014 0.037 0.014 0 100 0.014 0.020 average 0.200 0.037 0.200 0 100 0.200 0.020 stdev 1.212 0.022 1.212 0 086 1.212 0.004 p (t-test) 0.850 0 908 0.835
Figure imgf000181_0001
Alpha- 1 -antitrypsin: sCr or UO
0 hr prior to AKI stage 24 hr prior to AKI stage 48 hr prior to AKI stage
Cohort 1 Cohort 2 Cohort 1 Cohort 2 Cohort 1 Cohort 2 median 3 150 2.460 3.150 2.240 3.150 1.680 average 3 383 2.733 3.383 2.341 3.383 2.216 stdev 1 482 1.286 1.482 1.105 1.482 1.258 p (t-test) 0.041 0.001 0.005 min 0 854 1.270 0.854 0.737 0.854 1.080 max 8 930 6.130 8.930 5.540 8.930 5.580 n (Samp) 356 23 356 25 356 13 n (Pat) 121 23 121 25 121 13 sCr only
Figure imgf000181_0002
Figure imgf000182_0001
Leukocyte elastase:
Figure imgf000182_0002
sCr only
Figure imgf000182_0003
UO only
0 hr prior to AKI stage 24 hr prior to AKI stage 48 hr prior to AKI stage
Cohort 1 Cohort 2 Cohort 1 Cohort 2 Cohort 1 Cohort 2 median 254.910 293.033 254 910 328.996 254.910 331.015 average 306.181 356.872 306 181 363.019 306.181 433.999 stdev 197.948 224.078 197 948 243.399 197.948 407.742 p (t-test) 0.324 0.252 0.053 min 1.849 117.281 1 849 32.619 1.849 112.301
Figure imgf000183_0001
Soluble tumor necrosis factor receptor superfamily member 6:
Figure imgf000183_0002
sCr only
Figure imgf000183_0003
Soluble tumor necrosis factor ligand superfamily member 6:
Figure imgf000184_0001
sCr only
Figure imgf000184_0002
Soluble intercellular adhesion molecule 2:
Figure imgf000185_0001
sCr only
Figure imgf000185_0002
Figure imgf000185_0003
Heat shock protein beta- 1 :
Figure imgf000186_0001
sCr only
Figure imgf000186_0002
UO only
0 hr prior to AKI stage 24 hr prior to AKI stage 48 hr prior to AKI stage
Cohort 1 Cohort 2 Cohort 1 Cohort 2 Cohort 1 Cohort 2 median 78.528 25.248 78.528 50.559 78.528 8.406 average 124.114 81.591 124.114 50.559 124.114 25.041 stdev 126.609 146.704 126.609 43.989 126.609 na p (t-test) 0.358 0.418 na min 8.406 9.813 8.406 19.454 8.406 25.041 max 683.847 469.182 683.847 81.664 683.847 25.041 n (Samp) 64 9 64 2 64 1 n (Pat) 46 9 46 2 46 1
Soluble epidermal growth factor receptor:
Figure imgf000187_0001
sCr only
Figure imgf000187_0002
UO only
0 hr prior to AKI stage 24 hr prior to AKI stage 48 hr prior to AKI stage
Cohor t l Cohon t 2 Cohort 1 Cohort 2 Cohort 1 Cohort 2 median 10541 051 13660 928 10541.051 13735. 459 10541 051 6765 100 average 11680 245 12735 345 11680.245 14394. 423 11680 245 11884 006 stdev 4430 173 2531 142 4430.173 4142. 066 4430 173 na p (t-test) 0 552 0. 090 na min 6765 100 9355 404 6765.100 7026. 751 6765 100 11884 006 max 24717 033 16778 200 24717.033 21272. 844 24717 033 11884 006 n (Samp) 27 7 27 11 27 1 n (Pat) 25 7 25 11 25 1
[0168] In the following tables, the ability to distinguish cohort 1 (subjects remaining in RIFLE 0 or R) from Cohort 2 (subjects progressing to RIFLE I or F) was determined using ROC analysis. Soluble p-selectin:
Cohort 1 v Cohort 2, adjudicated on serum creatinine measurements or urine output
Figure imgf000188_0001
Cohort 1 v Cohort 2, adjudicated on serum creatinine
Figure imgf000188_0002
Cohort 1 v Cohort 2, adjudicated on urine output
Figure imgf000188_0003
Protein NOV homolog:
Cohort 1 v Cohort 2, adjudicated on serum creatinine measurements or urine output
Figure imgf000188_0004
Cohort 1 v Cohort 2, adjudicated on serum creatinine
Figure imgf000189_0001
Cohort 1 v Cohort 2, adjudicated on urine output
Figure imgf000189_0002
Netrin 4:
Cohort 1 v Cohort 2, adjudicated on serum creatinine measurements or urine output
Figure imgf000189_0003
Cohort 1 v Cohort 2, adjudicated on serum creatinine
Figure imgf000189_0004
Cohort 1 v Cohort 2, adjudicated on urine output
Figure imgf000189_0005
Alpha- 1 -antitrypsin:
Cohort 1 v Cohort 2, adjudicated on serum creatinine measurements or urine output
Figure imgf000190_0001
Cohort 1 v Cohort 2, adjudicated on serum creatinine
Figure imgf000190_0002
Cohort 1 v Cohort 2, adjudicated on urine output
Figure imgf000190_0003
Leukocyte elastase:
Cohort 1 v Cohort 2, adjudicated on serum creatinine measurements or urine output
Figure imgf000190_0004
Cohort 1 v Cohort 2, adjudicated on serum creatinine
Figure imgf000191_0001
Cohort 1 v Cohort 2, adjudicated on urine output
Figure imgf000191_0002
Soluble tumor necrosis factor receptor superfamily member 6:
Cohort 1 v Cohort 2, adjudicated on serum creatinine measurements or urine output
Figure imgf000191_0003
Cohort 1 v Cohort 2, adjudicated on serum creatinine
Figure imgf000191_0004
Cohort 1 v Cohort 2, adjudicated on urine output
Figure imgf000191_0005
Soluble tumor necrosis factor ligand superfamily member 6:
Cohort 1 v Cohort 2, adjudicated on serum creatinine measurements or urine output
Figure imgf000192_0001
Cohort 1 v Cohort 2, adjudicated on serum creatinine
Figure imgf000192_0002
Cohort 1 v Cohort 2, adjudicated on urine output
Figure imgf000192_0003
Soluble intercellular adhesion molecule 2:
Cohort 1 v Cohort 2, adjudicated on serum creatinine measurements or urine output
Figure imgf000192_0004
Cohort 1 v Cohort 2, adjudicated on serum creatinine
Figure imgf000193_0001
Cohort 1 v Cohort 2, adjudicated on urine output
Figure imgf000193_0002
Heat shock protein beta- 1 :
Cohort 1 v Cohort 2, adjudicated on serum creatinine measurements or urine output
Figure imgf000193_0003
Cohort 1 v Cohort 2, adjudicated on serum creatinine
Figure imgf000193_0004
Cohort 1 v Cohort 2, adjudicated on urine output
Figure imgf000193_0005
Soluble epidermal growth factor receptor:
Cohort 1 v Cohort 2, adjudicated on serum creatinine measurements or urine output
Figure imgf000194_0001
Cohort 1 v Cohort 2, adjudicated on serum creatinine
Figure imgf000194_0002
Cohort 1 v Cohort 2, adjudicated on urine output
Figure imgf000194_0003
[0169] Various threshold (or "cutoff) concentrations were selected, and the associated sensitivity and specificity for distinguishing cohort 1 from cohort 2, as shown in the following tables. OR is the odds ratio calculated for the particular cutoff concentration, and 95% CI is the confidence interval for the odds ratio. Soluble p-selectin: sCr or UO
Figure imgf000195_0001
sCr onl
Figure imgf000195_0002
Figure imgf000196_0001
Figure imgf000196_0002
Protein NOV homolog: sCr or UO
Figure imgf000196_0003
Figure imgf000197_0002
sCr onl
Figure imgf000197_0003
Figure imgf000197_0001
Figure imgf000197_0004
Figure imgf000198_0001
Netrin 4: sCr or UO
Figure imgf000198_0002
sCr only
Figure imgf000198_0003
Figure imgf000199_0001
Figure imgf000199_0002
Alpha- 1 -antitrypsin:
sCr or UO
Figure imgf000200_0001
sCr only
Figure imgf000200_0002
Figure imgf000201_0001
Leukocyte elastase: sCr or UO
Figure imgf000201_0002
Figure imgf000202_0002
sCr only
Figure imgf000202_0003
Figure imgf000202_0001
Figure imgf000202_0004
563.8166047 17% 90%
Soluble tumor necrosis factor receptor superfamily member 6: sCr or UO
Figure imgf000203_0001
sCr onl
Figure imgf000203_0002
Figure imgf000204_0001
Figure imgf000204_0002
Soluble tumor necrosis factor ligand superfamily member 6: sCr or UO
Figure imgf000205_0001
sCr only
Figure imgf000205_0002
Figure imgf000206_0001
Soluble intercellular adhesion molecule 2: sCr or UO
Figure imgf000206_0002
Figure imgf000207_0002
sCr only
Figure imgf000207_0003
Figure imgf000207_0001
Figure imgf000207_0004
783.804878 0% 91%
Heat shock protein beta- 1 : sCr or UO
Figure imgf000208_0001
sCr only
Figure imgf000208_0002
Figure imgf000209_0001
Soluble epidermal growth factor receptor: sCr or UO
Figure imgf000209_0002
na na sCr only
Figure imgf000210_0002
Figure imgf000210_0001
Figure imgf000210_0003
[0170] Example 11. Kidney injury markers for evaluating renal status in patients progressing from Stage R to Stages I and F
[0171] Patients were classified and analyzed as described in Example 9, but only those patients that reached Stage R were included in this example. Cohort 1 contained patients that reached stage R but did not progress to stage I or F within 10 days, and Cohort 2 included only patients that progressed to stage I or F. Marker concentrations in the plasma component from blood samples collected within 12 hours of reaching stage R were included in the analysis for both Cohort 1 and 2.
[0172] The following descriptive statistics were obtained Alpha- 1 -antitrypsin:
Figure imgf000211_0001
sCr only
Figure imgf000211_0002
UO only
0 hr prior to AKI stage 24 hr prior to AKI stage 48 hr prior to AKI stage
Cohort 1 Cohort 2 Cohort 1 Cohort 2 Cohort 1 Cohort 2 median 2.250 2.070 2.250 2.070 2.250 2.070 average 2.526 2.342 2.526 2.342 2.526 2.342 stdev 0.977 1.282 0.977 1.282 0.977 1.282 p (t-test) 0.635 0.635 0.635 min 1.310 0.748 1.310 0.748 1.310 0.748 max 4.900 5.090 4.900 5.090 4.900 5.090
Figure imgf000212_0001
Leukocyte elastase:
Figure imgf000212_0002
sCr only
Figure imgf000212_0003
Soluble tumor necrosis factor receptor superfamily member 6:
Figure imgf000213_0001
sCr only
Figure imgf000213_0002
UO only
0 hr prior to AKI stage 24 hr prior to AKI stage 48 hr prior to AKI stage
Cohort 1 Cohort 2 Cohort 1 Cohort 2 Cohort 1 Cohort 2 median 14241.486 11083.757 14241.486 11083.757 14241.486 11083.757 average 13099.883 11634.034 13099.883 11634.034 13099.883 11634.034 stdev 4274.913 3817.593 4274.913 3817.593 4274.913 3817.593 p (t-test) 0.441 0.441 0.441 min 6747.826 6493.109 6747.826 6493.109 6747.826 6493.109 max 18935.447 17259.343 18935.447 17259.343 18935.447 17259.343 n (Samp) 9 10 9 10 9 10 n (Pat) 9 10 9 10 9 10
[0173] In the following tables, the ability to distinguish cohort 1 (subjects remaining in RIFLE R) from Cohort 2 (subjects progressing to RIFLE I or F) was determined using ROC analysis. Alpha- 1 -antitrypsin:
Cohort 1 v Cohort 2, adjudicated on serum creatinine measurements or urine output
Figure imgf000214_0001
Cohort 1 v Cohort 2, adjudicated on serum creatinine
Figure imgf000214_0002
Cohort 1 v Cohort 2, adjudicated on urine output
Figure imgf000214_0003
Leukocyte elastase:
Cohort 1 v Cohort 2, adjudicated on serum creatinine measurements or urine output
Figure imgf000214_0004
Cohort 1 v Cohort 2, adjudicated on serum creatinine
Figure imgf000215_0001
Cohort 1 v Cohort 2, adjudicated on urine output
Figure imgf000215_0002
Soluble tumor necrosis factor receptor superfamily member 6:
Cohort 1 v Cohort 2, adjudicated on serum creatinine measurements or urine output
Figure imgf000215_0003
Cohort 1 v Cohort 2, adjudicated on serum creatinine
Figure imgf000215_0004
Cohort 1 v Cohort 2, adjudicated on urine output
Figure imgf000215_0005
[0174] Various threshold (or "cutoff) concentrations were selected, and the associated sensitivity and specificity for distinguishing cohort 1 from cohort 2, as shown in the following tables. OR is the odds ratio calculated for the particular cutoff concentration, and 95% CI is the confidence interval for the odds ratio:
Alpha- 1 -antitrypsin: sCr or UO
Figure imgf000216_0001
sCr only
Figure imgf000216_0002
Figure imgf000217_0001
Figure imgf000217_0002
Leukocyte elastase: sCr or UO
Figure imgf000217_0003
Figure imgf000218_0002
sCr only
Figure imgf000218_0003
Figure imgf000218_0001
Figure imgf000218_0004
Soluble tumor necrosis factor receptor superfamily member 6: sCr or UO
Figure imgf000219_0001
sCr only
Figure imgf000219_0002
Figure imgf000220_0001
[0175] While the invention has been described and exemplified in sufficient detail for those skilled in this art to make and use it, various alternatives, modifications, and improvements should be apparent without departing from the spirit and scope of the invention. The examples provided herein are representative of preferred embodiments, are exemplary, and are not intended as limitations on the scope of the invention. Modifications therein and other uses will occur to those skilled in the art. These modifications are encompassed within the spirit of the invention and are defined by the scope of the claims.
[0176] It will be readily apparent to a person skilled in the art that varying substitutions and modifications may be made to the invention disclosed herein without departing from the scope and spirit of the invention.
[0177] All patents and publications mentioned in the specification are indicative of the levels of those of ordinary skill in the art to which the invention pertains. All patents and publications are herein incorporated by reference to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference.
[0178] The invention illustratively described herein suitably may be practiced in the absence of any element or elements, limitation or limitations which is not specifically disclosed herein. Thus, for example, in each instance herein any of the terms "comprising", "consisting essentially of and "consisting of may be replaced with either of the other two terms. The terms and expressions which have been employed are used as terms of description and not of limitation, and there is no intention that in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention claimed. Thus, it should be understood that although the present invention has been specifically disclosed by preferred embodiments and optional features, modification and variation of the concepts herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention as defined by the appended claims.
[0179] Other embodiments are set forth within the following claims.

Claims

We claim:
1. A method for evaluating renal status in a subject, comprising: performing one or more assays configured to detect a kidney injury marker selected from the group consisting of soluble p-selectin, protein NOV homolog, soluble epidermal growth factor receptor, netrin-4, haptoglobin, heat shock protein beta-1, alpha- 1 -antitrypsin, leukocyte elastase, soluble tumor necrosis factor receptor superfamily member 6, soluble tumor necrosis factor ligand superfamily member 6, soluble intercellular adhesion molecule
2, active caspase-3, and soluble platelet endothelial cell adhesion molecule on a body fluid sample obtained from the subject to provide one or more assay results; and correlating the assay result(s) to the renal status of the subject.
2. A method according to claim 1, wherein said correlation step comprises correlating the assay result(s) to one or more of risk stratification, diagnosis, staging, prognosis, classifying and monitoring of the renal status of the subject.
3. A method according to claim 1, wherein said correlating step comprises assigning a likelihood of one or more future changes in renal status to the subject based on the assay result(s).
4. A method according to claim 3, wherein said one or more future changes in renal status comprise one or more of a future injury to renal function, future reduced renal function, future improvement in renal function, and future acute renal failure (ARF).
5. A method according to claim 4, wherein said assay result(s) comprise one or more of: (i) a measured concentration of soluble p-selectin,
(ii) a measured concentration of protein NOV homolog,
(iii) a measured concentration of soluble epidermal growth factor receptor,
(iv) a measured concentration of netrin-4,
(v) a measured concentration of haptoglobin,
(vi) a measured concentration of alpha- 1 -antitrypsin,
(vii) a measured concentration of leukocyte elastase,
(viii) a measured concentration of soluble tumor necrosis factor receptor superfamily member 6, (ix) a measured concentration of soluble tumor necrosis factor ligand superfamily member 6,
(x) a measured concentration of heat shock protein beta- 1 ,
(xi) a measured concentration of soluble intercellular adhesion molecule 2,
(xii) a measured concentration of active caspase-3, and
(xiii) a measured concentration of soluble platelet endothelial cell adhesion molecule, and said correlation step comprises, for each assay result, comparing said measure concentration to a threshold concentration, and for a positive going marker, assigning an increased likelihood of suffering a future injury to renal function, future reduced renal function, future ARF, or a future improvement in renal function to the subject when the measured concentration is above the threshold, relative to a likelihood assigned when the measured concentration is below the threshold or assigning a decreased likelihood of suffering a future injury to renal function, future reduced renal function, future ARF, or a future improvement in renal function to the subject when the measured concentration is below the threshold, relative to a likelihood assigned when the measured concentration is above the threshold, or for a negative going marker, assigning an increased likelihood of suffering a future injury to renal function, future reduced renal function, future ARF, or a future improvement in renal function to the subject when the measured concentration is below the threshold, relative to a likelihood assigned when the measured concentration is above the threshold or assigning a decreased likelihood of suffering a future injury to renal function, future reduced renal function, future ARF, or a future improvement in renal function to the subject when the measured concentration is above the threshold, relative to a likelihood assigned when the measured concentration is below the threshold.
6. A method according to claim 3, wherein said one or more future changes in renal status comprise a clinical outcome related to a renal injury suffered by the subject.
7. A method according to claim 1, wherein said assay result(s) comprise one or more of: (i) a measured concentration of soluble p-selectin,
(ii) a measured concentration of protein NOV homolog,
(iii) a measured concentration of soluble epidermal growth factor receptor, (iv) a measured concentration of netrin-4,
(v) a measured concentration of haptoglobin,
(vi) a measured concentration of alpha- 1 -antitrypsin,
(vii) a measured concentration of leukocyte elastase,
(viii) a measured concentration of soluble tumor necrosis factor receptor superfamily member 6,
(ix) a measured concentration of soluble tumor necrosis factor ligand superfamily member 6,
(x) a measured concentration of heat shock protein beta- 1 ,
(xi) a measured concentration of soluble intercellular adhesion molecule 2,
(xii) a measured concentration of active caspase-3, and
(xiii) a measured concentration of soluble platelet endothelial cell adhesion molecule, and said correlation step comprises, for each assay result, comparing said measure concentration to a threshold concentration, and for a positive going marker, assigning an increased likelihood of subsequent acute kidney injury, worsening stage of AKI, mortality, need for renal replacement therapy, need for withdrawal of renal toxins, end stage renal disease, heart failure, stroke, myocardial infarction, or chronic kidney disease to the subject when the measured concentration is above the threshold, relative to a likelihood assigned when the measured concentration is below the threshold, or assigning a decreased likelihood of subsequent acute kidney injury, worsening stage of AKI, mortality, need for renal replacement therapy, need for withdrawal of renal toxins, end stage renal disease, heart failure, stroke, myocardial infarction, or chronic kidney disease to the subject when the measured concentration is below the threshold, relative to a likelihood assigned when the measured concentration is above the threshold, or for a negative going marker, assigning an increased likelihood of subsequent acute kidney injury, worsening stage of AKI, mortality, need for renal replacement therapy, need for withdrawal of renal toxins, end stage renal disease, heart failure, stroke, myocardial infarction, or chronic kidney disease to the subject when the measured concentration is below the threshold, relative to a likelihood assigned when the measured concentration is above the threshold, or assigning a decreased likelihood of subsequent acute kidney injury, worsening stage of AKI, mortality, need for renal replacement therapy, need for withdrawal of renal toxins, end stage renal disease, heart failure, stroke, myocardial infarction, or chronic kidney disease to the subject when the measured concentration is above the threshold, relative to a likelihood assigned when the measured concentration is below the threshold.
8. A method according to claim 3, wherein the likelihood of one or more future changes in renal status is that an event of interest is more or less likely to occur within 30 days of the time at which the body fluid sample is obtained from the subject.
9. A method according to claim 8, wherein the likelihood of one or more future changes in renal status is that an event of interest is more or less likely to occur within a period selected from the group consisting of 21 days, 14 days, 7 days, 5 days, 96 hours, 72 hours, 48 hours, 36 hours, 24 hours, and 12 hours.
10. A method according to claim 1, wherein the subject is selected for evaluation of renal status based on the pre-existence in the subject of one or more known risk factors for prerenal, intrinsic renal, or postrenal ARF.
11. A method according to claim 1 , wherein the subject is selected for evaluation of renal status based on an existing diagnosis of one or more of congestive heart failure, preeclampsia, eclampsia, diabetes mellitus, hypertension, coronary artery disease, proteinuria, renal insufficiency, glomerular filtration below the normal range, cirrhosis, serum creatinine above the normal range, sepsis, injury to renal function, reduced renal function, or ARF, or based on undergoing or having undergone major vascular surgery, coronary artery bypass, or other cardiac surgery, or based on exposure to NSAIDs, cyclosporines, tacrolimus, aminoglycosides, foscarnet, ethylene glycol, hemoglobin, myoglobin, ifosfamide, heavy metals, methotrexate, radiopaque contrast agents, or strep tozotocin.
12. A method according to claim 1, wherein said correlating step comprises assigning a diagnosis of the occurrence or nonoccurrence of one or more of an injury to renal function, reduced renal function, or ARF to the subject based on the assay result(s).
13. A method according to claim 12, wherein said assay result(s) comprise one or more of:
(i) a measured concentration of soluble p-selectin, (ii) a measured concentration of protein NOV homolog,
(iii) a measured concentration of soluble epidermal growth factor receptor,
(iv) a measured concentration of netrin-4,
(v) a measured concentration of haptoglobin,
(vi) a measured concentration of alpha- 1 -antitrypsin,
(vii) a measured concentration of leukocyte elastase,
(viii) a measured concentration of soluble tumor necrosis factor receptor superfamily member 6,
(ix) a measured concentration of soluble tumor necrosis factor ligand superfamily member 6,
(x) a measured concentration of heat shock protein beta- 1 ,
(xi) a measured concentration of soluble intercellular adhesion molecule 2,
(xii) a measured concentration of active caspase-3, and
(xiii) a measured concentration of soluble platelet endothelial cell adhesion molecule, and said correlation step comprises, for each assay result, comparing said measure concentration to a threshold concentration, and for a positive going marker, assigning the occurrence of an injury to renal function, reduced renal function, or ARF to the subject when the measured concentration is above the threshold, or assigning the nonoccurrence of an injury to renal function, reduced renal function, or ARF to the subject when the measured concentration is below the threshold, or for a negative going marker, assigning the occurrence of an injury to renal function, reduced renal function, or ARF to the subject when the measured concentration is below the threshold, or assigning the nonoccurrence of an injury to renal function, reduced renal function, or ARF to the subject when the measured concentration is above the threshold.
14. A method according to claim 1, wherein said correlating step comprises assessing whether or not renal function is improving or worsening in a subject who has suffered from an injury to renal function, reduced renal function, or ARF based on the assay result(s).
15. A method according to claim 14, wherein said assay result(s) comprise one or more of: (i) a measured concentration of soluble p-selectin,
(ii) a measured concentration of protein NOV homolog,
(iii) a measured concentration of soluble epidermal growth factor receptor,
(iv) a measured concentration of netrin-4,
(v) a measured concentration of haptoglobin,
(vi) a measured concentration of alpha- 1 -antitrypsin,
(vii) a measured concentration of leukocyte elastase,
(viii) a measured concentration of soluble tumor necrosis factor receptor superfamily member 6,
(ix) a measured concentration of soluble tumor necrosis factor ligand superfamily member 6,
(x) a measured concentration of heat shock protein beta- 1 ,
(xi) a measured concentration of soluble intercellular adhesion molecule 2,
(xii) a measured concentration of active caspase-3, and
(xiii) a measured concentration of soluble platelet endothelial cell adhesion molecule, and said correlation step comprises, for each assay result, comparing said measure concentration to a threshold concentration, and for a positive going marker, assigning a worsening of renal function to the subject when the measured concentration is above the threshold, or assigning an improvement of renal function when the measured concentration is below the threshold, or for a negative going marker, assigning a worsening of renal function to the subject when the measured concentration is below the threshold, or assigning an improvement of renal function when the measured concentration is above the threshold.
16. A method according to claim 1, wherein said method is a method of diagnosing the occurrence or nonoccurrence of an injury to renal function in said subject.
17. A method according to claim 1, wherein said method is a method of diagnosing the occurrence or nonoccurrence of reduced renal function in said subject.
18. A method according to claim 1, wherein said method is a method of diagnosing the occurrence or nonoccurrence of acute renal failure in said subject.
19. A method according to claim 1, wherein said method is a method of diagnosing the occurrence or nonoccurrence of a need for renal replacement therapy in said subject.
20. A method according to claim 1, wherein said method is a method of diagnosing the occurrence or nonoccurrence of a need for renal transplantation in said subject.
21. A method according to claim 1, wherein said method is a method of assigning a risk of the future occurrence or nonoccurrence of an injury to renal function in said subject.
22. A method according to claim 1, wherein said method is a method of assigning a risk of the future occurrence or nonoccurrence of reduced renal function in said subject.
23. A method according to claim 1, wherein said method is a method of assigning a risk of the future occurrence or nonoccurrence of acute renal failure in said subject.
24. A method according to claim 1, wherein said method is a method of assigning a risk of the future occurrence or nonoccurrence of a need for renal replacement therapy in said subject.
25. A method according to claim 1, wherein said method is a method of assigning a risk of the future occurrence or nonoccurrence of a need for renal transplantation in said subject.
26. A method according to claim 5, wherein said one or more future changes in renal status comprise one or more of a future injury to renal function, future reduced renal function, future improvement in renal function, and future acute renal failure (ARF) within 72 hours of the time at which the body fluid sample is obtained.
27. A method according to claim 5, wherein said one or more future changes in renal status comprise one or more of a future injury to renal function, future reduced renal function, future improvement in renal function, and future acute renal failure (ARF) within 48 hours of the time at which the body fluid sample is obtained.
28. A method according to claim 5, wherein said one or more future changes in renal status comprise one or more of a future injury to renal function, future reduced renal function, future improvement in renal function, and future acute renal failure (ARF) within 72 hours of the time at which the body fluid sample is obtained.
29. A method according to claim 5, wherein said one or more future changes in renal status comprise one or more of a future injury to renal function, future reduced renal function, future improvement in renal function, and future acute renal failure (ARF) within 48 hours of the time at which the body fluid sample is obtained.
30. A method according to claim 5, wherein said one or more future changes in renal status comprise one or more of a future injury to renal function, future reduced renal function, future improvement in renal function, and future acute renal failure (ARF) within 24 hours of the time at which the body fluid sample is obtained.
31. Use of one or more kidney injury markers selected from the group consisting of soluble p-selectin, protein NOV homolog, soluble epidermal growth factor receptor, netrin- 4, haptoglobin, heat shock protein beta-1, alpha-1-antitrypsin, leukocyte elastase, soluble tumor necrosis factor receptor superfamily member 6, soluble tumor necrosis factor ligand superfamily member 6, soluble intercellular adhesion molecule 2, active caspase-3, and soluble platelet endothelial cell adhesion molecule for the evaluation of renal injury.
32. Use of one or more kidney injury markers selected from the group consisting of soluble p-selectin, protein NOV homolog, soluble epidermal growth factor receptor, netrin- 4, haptoglobin, heat shock protein beta-1, alpha-1-antitrypsin, leukocyte elastase, soluble tumor necrosis factor receptor superfamily member 6, soluble tumor necrosis factor ligand superfamily member 6, soluble intercellular adhesion molecule 2, active caspase-3, and soluble platelet endothelial cell adhesion molecule for the evaluation of acute renal injury.
33. A method according to claim 7, wherein the increased or decreased likelihood of subsequent acute kidney injury, worsening stage of AKI, mortality, need for renal replacement therapy, need for withdrawal of renal toxins, end stage renal disease, heart failure, stroke, myocardial infarction, or chronic kidney disease assigned to the subject is a likelihood that an event of interest is more or less likely to occur within 30 days of the time at which the body fluid sample is obtained from the subject.
34. A method according to claim 7, wherein the increased or decreased likelihood of subsequent acute kidney injury, worsening stage of AKI, mortality, need for renal replacement therapy, need for withdrawal of renal toxins, end stage renal disease, heart failure, stroke, myocardial infarction, or chronic kidney disease assigned to the subject is a likelihood that an event of interest is more or less likely to occur within 72 hours of the time at which the body fluid sample is obtained from the subject.
35. A method according to claim 7, wherein the increased or decreased likelihood of subsequent acute kidney injury, worsening stage of AKI, mortality, need for renal replacement therapy, need for withdrawal of renal toxins, end stage renal disease, heart failure, stroke, myocardial infarction, or chronic kidney disease assigned to the subject is a likelihood that an event of interest is more or less likely to occur within 24 hours of the time at which the body fluid sample is obtained from the subject.
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JP2011525260A JP2012501456A (en) 2008-08-28 2009-08-28 Methods and compositions for diagnosis and prognosis of renal injury and renal failure
MX2013010617A MX341926B (en) 2008-08-28 2009-08-28 Methods and compositions for diagnosis and prognosis of renal injury and renal failure.
BRPI0917711A BRPI0917711A2 (en) 2008-08-28 2009-08-28 method for assessing renal status in a patient, and use of one or more markers of renal injury
CN200980140805.3A CN102187220B (en) 2008-08-28 2009-08-28 For the method and composition of diagnosis and prognosis injury of kidney and kidney failure
EP09810695.8A EP2324355B1 (en) 2008-08-28 2009-08-28 Methods and compositions for diagnosis and prognosis of renal injury and renal failure
AU2009285550A AU2009285550B2 (en) 2008-08-28 2009-08-28 Methods and compositions for diagnosis and prognosis of renal injury and renal failure
US13/061,413 US11150250B2 (en) 2008-08-28 2009-08-28 Methods for diagnosing acute kidney injury or renal failure
CA2735587A CA2735587A1 (en) 2008-08-28 2009-08-28 Methods and compositions for diagnosis and prognosis of renal injury and renal failure
HK11107251.6A HK1153268A1 (en) 2008-08-28 2011-07-12 Methods and compositions for diagnosis and prognosis of renal injury and renal failure
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